Nusantara Bioscience vol. 5, no. 1, May 2013

| Nus Biosci | vol. 5 | no. 1 | pp. 1‐49 | May 2013 || ISSN 2087‐3948 | E‐ISSN 2087‐3956 |

Xenia macrospiculata ph

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Sebelas Maret University Surakarta

| Nus Biosci | vol. 5 | no. 1 | pp. 1‐49 | May 2013 |

| ISSN 2087‐3948 | E‐ISSN 2087‐3956 |

I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

ISSN: 2087-3948 Vol. 5, No. 1, pp. 1-7 E-ISSN: 2087-3956 May 2013

Macro-fungal diversity and nutrient content of some edible mushrooms of Nagaland, India

RAJESH KUMAR1,♥, ASHWANI TAPWAL2, SHAILESH PANDEY1, RAJIB KUMAR BORAH1, DEVAPOD BORAH1, JAYASREE BORGOHAIN1

1Rain Forest Research Institute, P.O. 136, Jorhat 785001, Assam, India. Tel.: +91-0376-2305106, ♥e-mail: [email protected] 2Forest Research Institute, Dehradun 248006, Uttrakhand, India

Manuscript received: 4 April 2013. Revision accepted: 2 May 2013.

Abstract. Kumar R, Tapwal A, Pandey S, Borah RK, Borah DP, Borgohain J. 2013. Macro-fungal diversity and nutrient content of some edible mushrooms of Nagaland, India. Nusantara Bioscience 5: 1-7. The northeast region of India abounds in forest wealth, including variety of flora and fauna. The high humidity during monsoon period provides ideal atmospheric conditions for the growth of diverse group of macrofungal fruit bodies. Nagaland, the northeastern state of India is rich in biodiversity and encompasses large numbers edible and non-edible mushroom species. Young and matured carpophores of 15 wild edible mushroom species were collected from 12 locations in different districts of Nagaland. Out of these four species belongs to family Agaricaceae, two belongs to Tricholomataceae and rest belongs to Boletaceae, Cantherallaceae, Russulaceae, Sarcoscyphaceae, Auriculariaceae, Polyporaceae, Schizophyllaceae, Pleurotaceae and Lyophyllaceae. The selected species were analyzed for proximate analysis of nutritional values. The protein content varies from 22.50-44.93% and carbohydrates were recorded 32.43-52.07% in selected species. The documentation of wild edible mushrooms is very scanty in Northeast India. The key objective of the present study was to generate a database on macrofungal diversity, ecology, ethnomycology, utilization and nutrient status of important wild edible mushroom species of Nagaland, which forms a part of the food culture of the native peoples.

Key words: Proximate analysis, carpophores, ethnomycology

Abstrak. Kumar R, Tapwal A, Pandey S, Borah RK, Borah DP, Borgohain J. 2013. Keanekaragaman makrofungi dan kandungan gizi beberapa jamur yang dapat dimakan dari Nagaland, India. Nusantara Bioscience 5: 1-7. Wilayah timur laut India memiliki kekayaan hutan yang berlimpah, termasuk berbagai spesies flora dan fauna. Kelembaban yang tinggi pada musim hujan memberikan kondisi udara yang ideal untuk pertumbuhan berbagai kelompok makrofungi. Nagaland, negara bagian India yang terletak di timur laut kaya akan keanekaragaman hayati, termasuk sejumlah besar spesies jamur yang dapat dan tidak dapat dimakan. Kaprofora muda dan masak dari 15 spesies jamur liar yang dapat dimakan dikumpulkan dari 12 lokasi di berbagai distrik di Nagaland. Dari jumlah tersebut, empat spesies milik keluarga Agaricaceae, dua milik Tricholomataceae dan sisanya milik Boletaceae, Cantherallaceae, Russulaceae, Sarcoscyphaceae, Auriculariaceae, Polyporaceae, Schizophyllaceae, Pleurotaceae dan Lyophyllaceae. Spesies terpilih dianalisis untuk mengetahui proksimat nilai gizi. Kadar protein bervariasi 22.50-44.93% dan karbohidrat tercatat 32.43-52.07% pada spesies yang dipilih. Dokumentasi jamur liar yang dapat dimakan sangat minim di timur laut India. Tujuan utama dari penelitian ini adalah untuk menghasilkan sebuah database tentang keragaman makrofungi, ekologi, etnomikologi, pemanfaatan dan status gizi spesies jamur liar penting yang dapat dimakan di Nagaland, yang merupakan bagian dari budaya makanan dari penduduk asli.

Kata kunci: Analisis proksimat, kaprofora, etnomikologi

INTRODUCTION

Mushrooms have been the objects of much curiosity and speculation since time immemorial. They are one of the most important components of the forest ecosystem. Their edibility, poisonous nature, psychotropic properties, mycorrhizal and parasitic associations with the forest trees make them economically important and interesting to study. The northeast region of India abounds in forest wealth, including many species of trees and other woody plants. The biodiversity of woody flora is correlated with an equally diverse mycoflora. The high humidity during monsoon period provides ideal atmospheric conditions for the growth of many saprophytes, including the mushrooms.

There are many mushrooms growing in the forests of Nagaland and local relish on them. They have diverse shapes, sizes and colors and also have varied appearance, ranging from patches on wood to brackets, coral-like tufts simple clubs rosettes cauliflower like structure or centrally or laterally stalked fruit bodies. Mushrooms can be categorized as edible or non-edible. The poisonous effects of mushrooms were dealt with in an epigram written by Euripides in about 450 B.C (Giovanni 1989). Right from the beginning man has learnt to differentiate the edible and non edible mushroom through numerous observation, trials and errors. Through these experiences man has learned to use mushrooms as a part of their diet. Seasonal mushroom hunting and collection are the part of seasonal activity of

5 (1): 1-7, May 2013 2

the people. Barros et al. (2008) reported the wild mushrooms are richer sources of protein and have a lower amount of fat than commercial mushrooms. The proteins of wild edible mushroom contains considerable amounts of non-essential amino acids like alanine, arginine, glycine, glutamic acid, aspartic acid, proline and serine (Manzi and Pizzoferrato 2000).The add-value arising from mushrooms are bioactive materials which lead to an increase in its consumption and therefore, stimulating the commercialization of edible species. Mushrooms also have been used extensively in traditional medicine for curing variety of diseases including viral infection, bacterial infection, cancer, tumor, inflammation, cardiovascular diseases (Benedict and Brady 1972; Iwalokum et al. 2007)

Many researchers have been working on wild mushroom and reported more than 2000 species of edible mushroom all over the world (Adhikari 2000; Purakasthya & Chandra 1985) have reported 283 edible species from India, out of which some are cultivated. Production of mushroom all over the world exceeds three million tones. Most of the exporting countries are Netherlands, Poland, Ireland, Belgium, India and China. Among these countries China is the largest exporter of preserved mushrooms. In India most commonly cultivated mushroom species are Button (Agaricus bisporus), Oyster (Pleurotus spp.) and Paddy straw mushroom (Volvariella volvacea) as documented by (Harsh and Joshi 2008).In India, mushroom is a unique non-traditional cash crop and as popular as food among the tribal people of north east India. Many rural communities of Nagaland are using mushrooms in their traditional dishes because of their delicious flavor. The favorable climatic condition of north-eastern states of India leads to rich mushroom diversity and form a valuable non-timber forest resource for local folk. Mushrooms are sold in traditional markets or commercially exploited as food or medicines (Tanti et al. 2011). Some of the edible species like Termitomyces eurrhizus, Lentinus conatus, Schizophyllum commune, Tricholoma giganteum and Pleurotus are sold in the markets of Kohima district of Nagaland by the local people (Tanti et al. 2011). In-spite of rich diversity of mushrooms in Nagaland state very few studies have been reported on diversity and market survey from North-Eastern Hills of India ( Verma et al. 1993; Singh et al. 2007 and Sarma et al. 2010).

The main objective of the present study was to generate a database on ecology, ethnomycology, utilization and nutrient status of important wild edible mushroom species of Nagaland, which forms a part of the food culture of the Nagaland people.

MATERIALS AND METHODS

Study area Nagaland is situated northeastern part of India having

longitude of 93°20´ E to 95°15´ E and Latitude 25°6´ N to 26°4´ N and having eleven districts with 16,579 Km2 area. The forest cover is about 86% including reserve forests. The prominent tribes of Nagaland are Chakhesang, Angami, Zeliang, Ao, Sangtam, Yimchunger, Chang, Sema, Lotha,

Khemungan, Rengma, Konyak, Pachury and Phom. The average annual rain fall ranges from 2000-2500 mm and average temperature during the summer ranges between 15 to 30ºC and in the winter it can fall below to 4ºC.

Sample collection and diversity analysis The periodic surveys were made to Lahorijan, Puliebzie,

Zakhama, Pherma, Mankoi, Chungtia, Nongkham, Namcha, and Tigit forest for the collection of macrofungi during rainy season (June to September) and winter (October to December) in 2010-2011. The collected samples were wrapped in wax paper and brought to the laboratory for identification and proximate analysis. The taxonomy has been worked on the basis of macro and microscopic characteristic following available literatures (Zoberi 1973; Alexopolous et al. 1996; Purakasthya 1985 and Singer 1986). The soft textured specimens were preserved in 2% formaldehyde and leathery textured were preserved in 4% formaldehyde and kept in museum of Forest Protection Division, Rain Forest Research Institute, Jorhat, Assam by assigning identification number. The traditional knowledge on the wild mushrooms were gathered from the local tribes and used to know the edibility and medicinal value. The frequency and density of different species has been determined by the following formulas:

No. of site in which the sp. is present Freq. of fungal sp. (%) = ----------------------------------------- x 100

Total no. of sites

Total no. of individual of a particular species Density = ------------------------------------------------------- x 100

Total no. of species

For proximate analysis, the fruit bodies were oven dried and powdered in a Moulinex blender. The fine powdered samples were stored in the desiccators and utilized for proximate and mineral nutrients analysis following Anthrone method (Fasidi and Kadiri 1993).

Moisture content: The fresh and oven dried weight (80°C for 48h) of each mushroom species was recorded moisture content was determined (Raghuramulu et al. 2003) by formula:

Fresh weigh – dry weight Moisture content (%) = -------------------------------- x 100 Fresh weigh Dry matter content: Weight obtained after oven drying

at 80°C for 48 h. Crude fiber: The Crude fibers content was calculated as

following equation: Crude fiber (g/100 g sample) = [100 − (moisture + fat)]

× (We-Wa)/Wt of sample (Raghuramulu et al. 2003). Protein content: 0.5 g of the powdered mushroom

sample was extracted with 50.0 cm of 2% NaCl in a water-bath at 60°C for 1 h. The extract was filtered out and 50.0 cm of 3% copper acetate monohydrate were added to the filtrate to precipitate protein. The precipitated protein was then centrifuged out and dissolves in 50 cm of 0.1 m NaOH. The quantity of protein in the alkaline solution was

KUMAR et al. -Macro-fungal diversity and nutrient content of some edible mushrooms 3

then determined using the folin-phenol method (Kadiri and Fasidi 1990).

Total carbohydrate estimation: The content of the available carbohydrate was determined by the following equation:

Carbohydrate (g/100 g sample) = 100-[(moisture + fat + protein + ash+ crude fiber) g/100 g] (Raghuramulu et al. 2003).

Ash content: The powdered mushroom sample (3.0 g)

was ashed in a Gallenkamp furnance in previously ignited and cooled crucible of known weight at 550°C for 6 h. Fairly cooled crucibles were put in desiccators and weighed (Raghuramulu et al. 2003). The ash content (g/100g) was calculated as following equation:

Weigh of ash Ash content (%) = -------------------------------- x 100 Weigh of sample taken Statistical Analysis: Experimental values are given as

means ± standard deviation (SD). Statistical significance was determined by one-way variance analysis (ANOVA). Differences at P < 0.05 were considered to be significant.

RESULTS AND DISCUSSION

The macroscopic characters like shape, size, colour, texture, attachment of stipe, smell, spore print, habit, and habitat has documented during the present study. The microscopic details like spore size, shape, colour and hyphal characteristics worked out in laboratory (Figure 1-2). The description of the collected specimens is recorded as follows:

Agaricus arvensis (Schaeff. ex Secr. s.). It grows on litter in the forest, cap 7-22 cm Convex to shield shaped creamy white or pale yellowish, stem 4-12 cm long; 1-2 cm thick; slightly bulbous and smooth, the ring is present with a double membrane, the lower splitting into a star-shape around the stem, gills Free from the stem; crowded, whitish to brown, spores ellipsoid, smooth, 7-8x4.5-5µ, spore print dark brown (Figure 1D, 2D).

Agaricus langei (Moller) Moller. It Grows on the ground, cap 4-12cm across, convex, densely covered in fine rust brown fibrous scales, gills pale fawny-pink at first becoming darker with age, stem 30-120x15-30mm, whitish with pink tinge and slightly mealy beneath the white pendulous ring, spores elliptic, 7-9x3.5-5µm, spore print purple-brown (Figure 1E, 2E).

Lepiota lilacea (Bres.). It Grows on the ground, cap 2-3.6 cm, convex, bell-shaped, dry, by maturity with a purple-brown to dark brown center and colored scales over a whitish to pinkish color, gills Free from the stem, white, close, stem 4-7 cm long, up to 5 mm thick, more or less equal, smooth, sheathing ring present, spores smooth; elliptical. Pileipellis hymeniform, 4-5x2-4 µm; spore print white (Figure 1L, 2L).

Lepiota magnispora (Murrill). Its growing scattered, gregariously, or in clusters in forest litter, cap 3-6 cm; convex to bell-shaped, dry; scaly; yellow to yellow-brown

or rusty brown with a darker, contrasting center, gills: Free from the stem; white; close, stem 3-8cm long,1.5 cm thick, more or less equal, with a slightly swollen base, hairy to shaggy with scattered brownish scales, spores smooth, dextrinoid, fusiform, with a convex curve on the adaxial side but not on the other side14-22x5-6.5 µm, spore print: dirty pink (Figure 1J, 2J).

Auricularia auricula-judae (St. Amans). It’s found on branches, fruit body 3-8 cm, gelatinous, ear shaped, outer surface brown with minute hairs, inner surface tan-brown, spore white, sausage shaped, 14-17×5-8µm, basidia cylindrical with three transverse septa, spore print white.Fig.1F, 2F).

Boletus aestivalis (Fr.). It Grows in woods, grows on the ground, fungus color white to cream, cap 7-20cm, pale straw-colour to pale snuff-brown, dry, soon becoming rough and cracking into small scales, particularly at centre, tubes white then greenish-yellow pores small, round, similarly colored, stem 60-150x20-50mm, robust, covered in a dense white network. Flesh white throughout, sometimes with slight yellowish tinges, spore subfusiform, 13-15x4.5-5.5 µm, spore print olivaceous snuff-brown (Figure 1O, 2O).

Cantharellus cibarius (Fr. Pfifferling). It grows in woods and on the ground, cap 3-5 inches wide convex at first with inrolled margin (edges), funnel shaped with a wavy margin with yellow orange color, the length of the stipe is similar to the width of the cap, gills are ridges that are forked and with blunt edges, the flesh is yellowish white, spore elliptical, 8-10x4.5-5.5µm, spore print white (Figure 1H, 2H).

Hypsizygus tessulatus (Bull. ex Fr.). It grows Singly or scattered on old hardwood trees, cap 2-5 cm; convex, flat at maturity, smooth; white to buff yellow, minutely hairy, stem is 4-24 cm, smooth, tapering towards the base, white hairs at the base, gills adnexed to sinuate, attached to the stem; nearly distant, cross-veined, spore globose, smooth, 4-5 µm (Figure 1C, 2C).

Pleurotus pulmonarius (Fr.) Quélet. It Growing on the tops of logs, Cap white to cream, 2-10cm, convex to flat, fan shaped in overlapping groups, very finely lined margin. Stem is Rudimentary, gills Whitish, cylindric, running down the stem; close or nearly distant; spore Cylindric, 7-10x2-4um, spore print White (Figure 1A, 2A).

Panus fulvus (Berk.). It grows on rotten wood of broad-leaved forest, fungus color white, cream or yellowish, pileus 4-9 cm, funnel-shaped, yellowish brown, tomentous, margin with strips, gills decur-rent, brown, stipe central, 3.5-5 × 0.4-0.6 cm, solid, brown, covered with ark brown hairs, spores elliptical, hyaline, smooth, I-, 6.5-7.5 × 2.5-3 μm; spore print white (Figure 1M, 2M).

Lactarius hygrophoroides (Berk. et Curt). Its grows in woods and on the ground, cap 3-10 cm; convex to shield shaped dusted with a whitish bloom; velvety; dry; the margin slightly rugged, stem 3-6 cm long; 0.6-1.4 cm thick; colored velvety like the cap, the length of the stipe is similar to the width of the cap, gills attached to the stem distant, the flesh is white, exudes watery latex, spores 7-9x5.5-7 µm; Macrocystidia absent, spore print is white (Figure 1I, 2I).

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Figure 1. Fruting body of collected mushrooms. A. Pleurotus pulmonarius, B. Schizophyllum commune, C. Hypsizygus tessulatus, D. Agaricus arvensis, E. Agaricus langei, F. Auricularia auricula-judae, G. Lepista irina, H. Cantharellus cibarius, I. Lactarius hygrophoroides, J. Lepiota magnispora, K. Cookeina sulcipes, L. Lepiota lilacea, M. Panus fulvus, N. Melanoleuca grammopodia, O. Boletus aestivalis

A B C

D E F

G H I

J K L

M N O


Figure 2. Spores of the collected mushrooms. A. Pleurotus pulmonarius, B. Schizophyllum commune, C. Hypsizygus tessulatus, D. Agaricus arvensis, E. Agaricus langei, F. Auricularia auricula-judae, G. Lepista irina, H. Cantharellus cibarius, I. Lactarius hygrophoroides, J. Lepiota magnispora, K. Cookeina sulcipes, L. Lepiota lilacea, M. Panus fulvus, N. Melanoleuca grammopodia, O. Boletus aestivalis

A B C

D E F

G H I

J K L

M N O

5 (1): 1-7, May 2013 6

Cookeina sulcipe (Berk.) Kuntze. It grows as saprotrophs on dead wood, fruiting bodies cup-shaped to funnel-shaped, brightly-colored, and yellow to red. The outer surface is less brightly colored the walls of the apothecium, is thin and flexible and has tiny hairs on the upper rim of the cup, asci are constricted abruptly below and form a blunt, rounded base with a slim, tail-like connection, ascospores ellipsoidal and smooth 20-40.5µm long (Figure 1K, 2K ).

Schizophyllum commune (Fr. Gemeiner Spaltblättling). It grows in dead wood of deciduous trees, fruiting body 1-5 cm wide, fan-shaped small hairs on the upper surface, white to grayish, stem rudimentary or absent, gills Under surface of the fruiting body composed of gill-like folds in the undersurface that are distinctively split, spore Cylindric, 5x3um, cystidia absent spore print White (Figure 1B, 2B).

Lepista irina (Fr.) H.E. It’s found in open woodland, cap light brown, 5-11cm across, flattened-convex, wavy at the margin, stem 55-97x8-20mm, dirty white, covered in long fibres, ochraceous near the base, gills emarginate, crowded, spores oval,7-9x3.5-4µm, spore print dirty pink (Figure 1G, 2G).

Melanoleuca grammopodia (Bull.) Murrill. It found in woods on leaf mulch and on composted soil, cap convex, then flattened, with a broad central bump, often depressed, smooth, gills broad, emarginate, whitish, or cream, stem equal with a broad base, whitish, with brown fibres along the length, spores ellipsoidal, smooth 8.5-9.5x 5-6 µm, basidia four spored, spore print white (Figure 1N, 2N).

Species diversity of macrofungi is related to the particular habitats. The factors like geographic location, elevation, temperature, humidity, light and surrounding flora greatly influence the growth and development of macrofungi. In present study, the fungal fruitbodies were collected from 12 different locations of nine districts of Nagaland. 15 species of edible mushrooms were found, out of which 4 belongs to family Agaricaceae, 2 belongs to Tricholomataceae, and one each in Boletaceae, Cantherallaceae, Russulaceae, Sarcoscyphaceae, Auricula-

riaceae, Polyporaceae, Schizophyllaceae, Pleurotaceae and Lyophyllaceae. The diversity analysis revealed that maximum frequency occurrence was exhibited by Auricularia auricula-judae (66.6%), followed by Agaricus langei and Lactarius hygrophoroides (58.3% each), Pleurotus pulmonarius (50%) and minimum (16.6%) by Melanoleuca grammopodia. The rest of species exhibited the frequency of distribution between 16.6-50%. All of the selected species are edible and among which four have medicinal importance also (Table 1). Recently, Tanti et al. (2011) has recorded 13 number of macrofungi under 9 genera and six families available in the market of Kohima town of the Nagaland.

Mushrooms are delicious food due their high quality protein, vitamins and minerals. The proximate composition of the selected edible mushroom species has been presented in Table 2. Fresh mushrooms contained about 90% moisture and 10% dry matter and dry mushrooms contained about 90% dry matter and 10% moisture (Chang and Buswell 1996). In the present study it was observed that the moisture content of the collected mushroom samples ranges from 52.11-95.13%. The Pleurotus, Agaricus and Lepiota have higher moisture content in comparison to other species. The dry matter content ranged from 2.1-4.2% with exception to S. commune, having 12.9% dry content. Crude fibres were recorded minimum for A. arvensis (0.14%) and maximum 12.9% for H. tessulatus, rest were in between. Edible mushrooms are highly valued as a good source of protein and their protein contents usually ranges from 28.93% to 39.1% of dry weight (Ragunathan et al. 2003; Sanmee et al. 2003). Following similar trend, the highest protein content was recorded for L. hygrophoroides (44.93%) and lowest for S. commune (22.50%). The carbohydrates content of edible mushrooms usually range from 40.6% to 53.3% of dry weight (Khanna et al. 1992; Ragunathan et al. 1996). In the present study, species have carbohydrates between 32.43-52.07%. The ash content has exhibited quite variation from 0.18-14.97% in different species.

Table 1. Frequency of occurrence and density of macrofungi

Name of the species Family Host/Substratum Use Freq. of occurr (%) Density ID

number Agaricus arvensis Agaricaceae Grows on litter Edible 25.0 66.6 NL-000256Agaricus langei Agaricaceae Grows on ground Edible 58.3 108.3 NL-000312Lepiota lilacea Agaricaceae On ground Edible 41.6 58.3 NL-000305Lepiota magnispora Agaricaceae Forest litter Edible 33.3 33.3 NL-000291Auricularia auricula-judae Auriculaceae Dead bamboo culm, Underwood Edible, medicine 66.6 133.3 NL-000270Boletus aestivalis Boletaceae On wood, ground Edible 25.0 25.0 NL-000280Cantharellus cibarius Cantharallaceae On live coconut/ Dead wood logs Edible 33.3 41.6 NL-000255Hypsizygus tessulatus Lyophyllaceae Old hardwood trees Edible, medicine 33.3 91.6 NL-000286Pleurotus pulmonarius Pleurotaceae Wood logs Edible 50.0 116.6 NL-000105Panus fulvus Polyporaceae Rotten wood Edible 33.3 33.3 NL-000172Lactarius hygrophoroides Russulaceae Wood, litter Edible, medicine 58.3 91.6 NL-000258Cookeina sulcipes Sarcoscyphaceae Dead wood Edible 25.0 50.0 NL-000307Schizophyllum commune Schizophyllaceae Dead wood of deciduous trees Edible, medicine 41.6 75.0 NL-000143Lepista irina Tricholomataceae Woodland Edible 50.0 58.3 NL-000290Melanoleuca grammopodia Tricholomataceae Leaf mulch, composted soil Edible 16.6 33.3 NL-000112


Table 2. Proximate composition (g/100g) of 15 selected wild edible mushroom species (mean ±SD) Mushrooms Moisture Dry matter Crude fibre Protein Carbohydrates Ash Agaricus arvensis 94.90±1.80 4.20±0.60 0.14±0.02 32.87±1.69 32.91±1.80 0.18±0.01 Agaricus langei 84.82±1.72 4.10±0.65 3.28±0.05 35.14±1.04 34.83±1.82 14.10±0.61 Lepiota lilacea 83.20±1.73 4.20±0.67 11.98±0.64 28.12±1.40 49.33±1.94 8.09±0.77 Lepiota magnispora 93.31±2.02 2.40±0.53 5.20±0.29 27.55±1.25 35.00±1.58 3.05±0.57 Auricularia auricula-judae 95.17±2.03 2.20±0.59 2.81±0.04 36.30±1.33 33.23±1.67 7.07±0.52 Boletus aestivalis 77.01±1.25 4.10±0.65 12.13±0.58 32.76±1.47 52.07±2.81 14.97±0.73 Cantharellus cibarius 87.82±1.63 2.20±0.51 1.40±0.28 34.17±1.26 47.00±2.24 7.78±0.63 Hypsizygus tessulatus 83.40±1.39 3.10±0.87 12.90±0.35 37.80±1.25 51.20±2.27 9.09±0.78 Pleurotus pulmonarius 95.13±1.83 3.90±0.64 4.12±0.64 37.63±1.24 43.40±2.15 10.17±1.26 Panus fulvus 52.11±1.14 2.10±0.63 6.08±0.52 27.06±1.62 33.04±1.28 3.11±0.47 Lactarius hygrophoroides 70.00±1.28 3.30±0.86 10.58±0.35 44.93±1.79 42.00±1.64 2.00±0.29 Cookeina sulcipes 88.48±1.51 2.30±0.58 0.16±0.02 28.93±1.65 50.20±2.38 6.55±0.58 Schizophyllum commune 87.30±1.29 12.90±1.74 6.50±0.67 22.50±0.67 32.43±1.21 10.10±1.14 Lepista irina 83.82±1.58 2.10±0.47 6.08±0.52 26.12±1.50 50.20±2.34 3.16±0.59 Melanoleuca grammopodia 67.34±1.89 3.10±0.84 8.12±0.64 36.27±1.52 33.04±1.43 4.13±0.68 CD (p < 0.05) 4.72 2.34 1.32 4.12 5.88 2.07

CONCLUSION

The identification and use of wild edible mushrooms play a vital role in enrichment of the socio-economic life of the tribal people. The current environmental issues of global warming and climate change would adversely affect the regeneration and growth pattern of the delicate fungi which requires a specific micro-climate. Consequently, the high nutritional quality and unique flavor of these mushrooms are likely to be lost if these wild edibles are not properly documented. However, a through screening is needed to delimit their different medicinal properties which will not only help in solving the food crisis which is prevalent in the rural poor population but will also add medicinal touch to their food.

ACKNOWLEDGEMENTS

The authors are gratefully acknowledged to Indian Council of Forestry Research and Education (ICFRE) for funding the research project: No-RFRI-39/2010-11/FP.

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Impact of rhizobial inoculation and nitrogen utilization in plant growth promotion of maize (Zea mays L.)

RAMESH K. SINGH1,♥, NAMRATA MALIK1,2, SURENDRA SINGH2 1 PGPR Laboratory, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, Uttar

Pradesh, India. Tel./fax.: +91-542-2368993, ♥e-mail: [email protected] 2Department of Botany, Faculty of Science, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India

Manuscript received: 2 April 2013. Revision accepted: 30 April 2013.

Abstract. Singh RK, Malik N, Singh S. 2013. Impact of rhizobial inoculation and nitrogen utilization in plant growth promotion of maize (Zea mays L.). Nusantara Bioscience 5: 8-14. During the course of growing population demands there has been an increasing interest in exploring the possibility of extending the beneficial interaction between cereals and plant growth promoting rhizobacteria (PGPR). Endophytes are a group of microorganism that resides mostly in the intercellular space of various parts of plants including cereals. Assessment of plant growth promoting properties of the five-rhizobial strains belonging to α subclass i.e. Rhizobium leguminosarum bv. phaseoli RRE6 and R. undicola RRE36 and those belonging to β subclass i.e. Burkholderia cepacia (RRE3, RRE5, RRE25) was done by growing maize plants inoculated with these strains. Inoculated maize plants showed a significant increase in plant height, root length, shoot and root dry weight over uninoculated control. R. leguminosarum bv. phaseoli RRE6 and B. cepacia RRE5 among the α and β-subclass representatives respectively, gave the best inoculation response. Effect of nitrate supplementation upon maize-RRE6 and RRE5 association was also studied and a significant increase in all the growth parameters and colonization ability was recorded when nitrate was present as a supplement over uninoculated control and maize-RRE6 and RRE5 in absence of external nitrate.

Key words: Rhizobium leguminosarum bv. phaseoli, R. undicola, B. cepacia, Zea mays, nitrate utilization

Abstrak. Singh RK, Malik N, Singh S. 2013. Pengaruh inokulasi rhizobia dan perlakuan nitrogen terhadap pertumbuhan tanaman jagung (Zea mays L.). Nusantara Bioscience 5: 8-14. Dalam penelitian pertumbuhan populasi telah terjadi peningkatan minat dalam mengeksplorasi kemungkinan mendapatkan keuntungan dari interaksi antara sereal dan pertumbuhan tanaman yang dibantu rhizobakteri (PGPR). Endofit adalah kelompok mikroorganisme yang umumnya berada di ruang antar sel berbagai bagian tanaman termasuk sereal. Penilaian sifat-sifat pertumbuhan tanaman yang dipromosikan oleh lima strain rhizobia sub kelas α, yaitu Rhizobium leguminosarum bv. phaseoli RRE6 dan R. undicola RRE36 serta sub kelas β, yaitu Burkholderia cepacia (RRE3, RRE5, RRE25) dilakukan dengan menumbuhkan tanaman jagung yang diinokulasi dengan strain-strain tersebut. Tanaman jagung yang diinokulasi menunjukkan peningkatan yang signifikan dalam tinggi tanaman, panjang akar, tunas dan bobot kering akar dibandingkan kontrol tanpa inokulasi. R. leguminosarum bv. phaseoli RRE6 dan B. cepacia RRE5 yang masing-masing secara berturut-turut mewakili sub kelas α dan β, memberikan respon inokulasi terbaik. Pengaruh suplementasi nitrat pada jagung yang berasosiasi dengan RRE6 dan RRE5 juga dipelajari dan peningkatan yang signifikan dalam semua parameter pertumbuhan dan kemampuan kolonisasi dicatat dimana nitrat hadir sebagai suplemen dibandingan kontrol tanpa inokulasi dan jagung-RRE6 dan RRE5 tanpa tambahan nitrat dari luar.

Kata kunci: Rhizobium leguminosarum bv. phaseoli, R. undicola, B. cepacia, Zea mays, pemupukan nitrat

INTRODUCTION

PGPR are plant growth promoting rhizobacteria that directly or indirectly induce beneficial effects on plant growth and development. They can occur naturally as rhizospheric, endophytic or symbiotic component of bacteria plant association. PGPR can affect plant growth and development either indirectly or directly. Direct mechanisms include production of phytohormones, synthesis of 1 aminocyclopropane-1-carboxylate (ACC) deaminase, phosphorous solubilization, nitrogen fixation and siderophore production. Some indirect mechanisms are that they act as biocontrol agents and induce systemic resistance in plants (Ashraf et al. 2013). Occurrence of endophytic association has been reported in many non

leguminous crops such as maize, wheat, millets, kallar grass, sugarcane, etc. (Webster et al. 1998, Chaintreuil et al. 2000; Gutierrez-Zamora and Martinez-Romero 2001; Matiru and Dakora 2004). Plant-microbe interactions may occur at phyllosphere, endosphere and rhizosphere (Bhattacharyya and Jha 2012).

Maize (Zea mays) is widely cultivated throughout the world and it is one of the most important staple grain crops in the world. Nitrogen and phosphorus are two of the essential nutrients for maize plant growth and development. Large quantities of chemical fertilizers are used as to replenish soil N and P. Use of high levels of nitrogenous fertilizers in crop production has its drawbacks. Only one-third of the nitrogen applied as a chemical fertilizer is used up by the crop. The non-assimilated nitrogen results in

SINGH et al. – Impact of rhizobium and nitrogen in growth of maize 9

nitrate (NO3-) contamination of ground water supplies

(Mytton 1993; Shrestha and Ladha 1998), a potential health hazard; soil acidification (Kennedy and Tchan 1992) and increased denitrification. Soil acidification and denitrification results in high emission of nitrous oxide (N2O), a potent greenhouse gas, which enhances global warming (Bronson et al. 1997). Thus we are in dire need of exploring alternate or supplementary non-polluting sources of N for agriculture (Ladha et al. 1997). This problem could be solved if maize and other cereals were able to establish more intimate associations with plant growth promoting microorganisms. It, therefore, would be a noteworthy achievement if maize could profit from biological nitrogen fixation thereby decreasing its requirement and dependence on chemical nitrogenous fertilizers (Chelius and Triplett 2000; 2001). We have therefore endeavoured to see if inoculation of some endophytic bacteria can improve growth performance of maize.


Details of bacterial strains used in the study are listed in Table 1. Spontaneos mutants of Burkholderia strains RRE3, RRE5, RRE25, and Rhizobium strains RRE6 and RRE36 were isolated which were resistant to various antibiotics to be used as genetic marker during the study.

Table 1. Bacterial strains used in the present study

Strains Accession number

Burkholderia cepacia RRE3 AY 946010 B. cepacia RRE25 EU 246850 B. cepacia RRE5 AY 946011 Rhizobium leguminosarum bv. phaseoli RRE6 AY 946012 R. undicola RRE36 EU 512923

Characterization of the strains For growth studies and utilization of different nitrogen

sources all the bacterial strains were inoculated in Yeast Extract Mannitol (YEM, Vincent 1970) and grown upto 109 cells ml-1 and Rhizobial Minimal Medium (RMM) for specific experiments (Diebold and Noel 1989). Sodium glutamate of minimal medium was replaced by nitrogen sources like sodium nitrite, sodium nitrate and ammonium sulphate (10mM each) to study nitrogen utilization. Indole acetic acid (IAA) quantification and siderophore production were assayed as described by Patten and Glick 2002; Penrose and Glick 2003. Phosphatase enzyme activity was studied using Pikovskaya’s broth medium. Pectinase assay was done according to the method of Mandels (1985). To estimate nitrogenase activity the acetylene reduction assay of Stewart et al. (1967) was used.

Plant growth experiment (green house conditions) To study the effect of inoculation with different

antibiotic marked endophytic strains on growth of maize

plant, seeds of the maize cultivar Malaviya 1 were selected and surface sterilized following standard protocol according to Singh et al. 2006. Three days old maize seedling, with root length ranging from 2.0 cm to 3.0 cm, were inoculated separately with one ml each of exponentially grown bacterial culture having cell population of 109 CFU (colony forming units) ml-1. Uninoculated seedlings served as control. The germinated maize seedlings were transferred aseptically to plastic pots containing sterile sand. Sand was washed three times with tap water and dried in hot air oven. Washed and dried sand was then autoclaved twice for 20 minutes at 120 °C with an interval of 24 hours. All treatments were arranged in 25 pots i.e. 5 replicates with 5 pots per replication. The plants were incubated in plant growth chamber under a combination of fluorescent and incandescent light with a light intensity of 16000 lux, with a cycle of 16 hrs light and 8 hrs dark cycle temperatures of 28°/23°C and relative humidity of 55/75%. Plants were regularly watered with Nitrogen free Fahraeus medium (NFM, Fahraeus 1987) and were harvested at 35 days after inoculation.

Re-isolation and characterization of putative endophytes

Root portion of uprooted maize plants were cut into small pieces of 5 mm length. Surface sterilization was done twice once with 95% ethanol and then with 0.2% acidified HgCl2. Sterilized root pieces were macerated in 10ml sterilized distilled and were decimally diluted (10-5) and spread on YEM agar plates supplemented with appropriate antibiotics.

Growth of maize seedling as influenced by nitrate availability and PGPR inoculation

Two antibiotic resistant derivatives of endophytic rhizobia, i.e., Rhizobium leguminosarum bv. phaseoli RRE6strR (resistant to streptomycin 100μg/ml) and Burkholderia cepacia RRE5strR (resistant to streptomycin 500μg/ml) were used for this study. Surface sterilized seeds of maize (Malaviya 1) were germinated in Petri plates containing moistened filter paper under aseptic conditions. Three-day old seedlings, with root length ranging from 2 to 4 cm, were selected and soaked in 25 ml of bacterial suspension of RRE-5strR and RRE-6strR for 90 min. Uninoculated seedlings served as control. Soaked seedlings were then removed and transferred to agar-water plates supplemented without and with 10mM NO3

-. Four seedlings were planted on each Petri plate and five replicates were considered for each treatment. These seedlings were allowed to grow for 48 h and then removed from agar water plates and washed in distilled water in order to remove the agar adhering to the roots. Shoot length, root length and fresh weight were recorded. Two grams of root were taken from each treatment separately and macerated. Macerate was decimally diluted, 100 μl of each of these solutions was spread on streptomycin-containing YEM plates. Subsequently, the plates were incubated at 28°C and CFU were counted. For analysis of data, the completely randomized design was used.

5 (1): 8-14, May 2013 10

Statistical analysis Data related to plant growth parameters were subjected

to analysis of variance using SPSS software. Treatment means were compared at 95% and 99% probability level (P = 0.05 and 0.01), and the same set of data was further analyzed to calculate the least significant difference at P = 0.05 and 0.01, respectively.


Response of bacterial inoculation on the growth of maize plant under green house conditions

The maize cultivar Malaviya 1 was used in plant growth experiment test to study the effect of inoculation of various endophytic rhizobial isolates on its growth. The statistical analysis showed that there was significant effect of inoculation over the control (uninoculated). Statistically significant differences were also observed among the isolates. Significant increase in plant height was observed in all rhizobial treatments (Figure 1). R. leguminosarum RRE6 gave the best inoculation response with maize and resulted in maximum increase in plant height, whereas B. cepacia RRE3 gave the minimum plant height increase over the control (Table 2). R. undicola RRE36 also showed a very good response to inoculation in terms of plant height. When strains were compared among themselves, B. cepacia RRE5, R. undiocola RRE36 and B. cepacia RRE3 differed significantly from each other.

Insofar as root length is concerned, it was found that there was significant increase over the control in the cases of R. leguminosarum RRE6, B. cepacia RRE5 and R. undicola RRE36. But in cases of B. cepacia strains RRE3 and RRE25, differences were statistically non significant (Table 2). In the case of RRE6, there was a significant increase compared to the control (60% increase) due to inoculation. RRE36 also showed a significant increase when compared with the control plant. Statistically significant difference was observed between both Burkholderia strains (RRE5 and RRE25); RRE5 was found

to be more effective. A significant increase in shoot dry weight was observed in maize plants inoculated with all endophytic rhizobial isolates. Maximum shoot dry weight was observed in plants inoculated with R. leguminosarum RRE6 followed by R. undicola RRE36 (Table 2). Shoot dry weight was found to be more in the case of Rhizobium strains when compared with Burkholderia strains.

A significant increase in root dry weight was exhibited by all the bacterial endophytes. The best performance for dry weight was shown by R. leguminosarum RRE6 followed by R. undicola RRE36. When Burkholderia

strains were considered, significant increase over the control was apparent (Table 2). The cell densities from the sterilized root macerates of uprooted maize plants were calculated and found to be similar in each case (Table 3).

Table 2. Effect of inoculation of endophytic rhizobia on promotion of growth of maize plant (green house conditions)

TreatmentPlant height in cm plant-1

Root length in cm plant-1

Shoot dry weight in g plant-1

Root dry weight in g plant-1

Control 35.97 23.6 0.060 0.051 RRE5 45.47*

(26.41) 28.62* (21.2)

0.167* (178)

0.079* (54.9)

RRE6 56.39* (56.76)

37.72* (59.8)

0.236* (293)

0.100* (96.07)

RRE25 47.20* (31.22)

24.17* (2.41)

0.166* (176)

0.074* (45.09)

RRE36 51.59* (43.42)

32.22* (36.52)

0.208* (246)

0.088* (72.5)

RRE3 40.80* (13.42)

23.87* (1.1)

0.107* (78.3)

0.068* (33.33)

CD at 5% 4.32 3.433 0.036 0.017 SEM ± 2.06 1.624 0.017 0.008 Note: RRE6 and RRE36 = Rhizobium strains; RRE5, RRE3 and RRE25= Burkholderia strains; CD = Critical difference; SEM = Standard error of mean (n = 4). Values in parentheses indicate % increase over control. *significant at 5% Table 3. Cell density of endophytic bacterial strains isolated from inoculated maize plants

Bacterial strains Cell density* (X 108cells ml-1)

RRE5 7.88±1.05 RRE3 8.93±0.09 RRE25 7.12±1.08 RRE6 8.26±0.09 RRE36 7.25±0.08 Control 0 Note: *Values are expressed as g-1 root fresh weight ml-1

Figure 1. Maize plants inoculated with endophytic bacterial isolates. Note: 1. Control (uninoculated), 2. R. undicola RRE36, 3. B. cepacia RRE3, 4. R. leguminosarum RRE6


Biological activity of the rhizobial endophytes All the 5 endophytes i.e. R. leguminosarum bv. phaseoli

RRE6, R. undicola RRE36, B. cepacia (RRE3, RRE5, RRE25) grew well in the presence of different nitrogen sources (Table 4). It was found that all the strains were similar in their ability to produce auxin, at an average of 3.00 μg ml-1 (Table 5). The strains were also able to solubilize phosphate and pectin (Figure 2a, b). Nitrogenase activity was found to similar in all the strains. The production of siderophores by bacterial endophytes was positive as detected on chrome azurol S (CAS) plates. Growth of all the strain was found to be identical in both rich medium and minimal medium.

From the present observations it was clear that among the α and β subclass representatives, R. leguminosarum RRE6 and B. cepacia RRE5 were the most effective in increasing different growth parameters of maize. The present results clearly indicates that the α-subclass representatives were better growth enhancer as compared to β-subclass ones, though representatives of both the classes showed significant increase in the growth when compared to the control or uninoculated plants. For further study on these aspects, B. cepacia RRE5 and R. leguminosarum RRE6 were used to compare between Burkholderia and Rhizobium.

Table 4. Utilization of nitrogen sources by rhizobial strains in minimal medium (MM)

Strain Absorbance at 420 nm (after 4 days of inoculation) MM+NO3 MM+NO2 MM+NH4

RRE3 1.75 1.68 0.66 RRE36 1.45 1.52 0.86 RRE25 1.91 1.85 0.84 RRE5 2.07 1.47 0.91 RRE6 2.25 1.77 0.98

Table 5. Biological activity of the rhizobial endophytes

Rhizobial strains

IAA production(μgml-1)

Siderophore production

Phosphate solubilization (mm h-1)

Pectinase activity (mm h-1)

Nitrogenase activity (nmol C2H4h-1mg-1 protein)

RRE5 3.11 ++ 0.14 0.10 22.75 RRE6 3.35 ++ 0.17 0.11 21.52 RRE3 2.17 ++ 0.09 0.10 21.27 RRE25 2.87 ++ 0.11 0.04 18.74 RRE36 3.01 ++ 0.10 0.05 19.75

Effect of nitrate availability and PGPR inoculation on the growth of maize seedlings

In this experiment, observations were recorded 48 h after inoculation. In the presence of nitrate, the growth of maize seedling was enhanced significantly upon inoculation with both the strains (Figure 3). The combined effect of nitrate enrichment and strains was much more significant than the individual effects of these two variables. Root length, shoot length and fresh weight significantly increased in all the cases when compared to the control (uninoculated) plants. Effect was more pronounced in R. leguminosarum RRE6 than in B. cepacia RRE5 when nitrate was added as supplement (Table 6).

Number of bacterial colonies recovered from root macerates of inoculated plants of maize

The number of endophytes recovered from the surface-sterilized roots in case of R. leguminosarum RRE6 was found to be 3.39 x 106, which was higher than B. cepacia RRE5 (2.35 x 106). In both the cases, nitrate addition increased the number of colonies and in case of RRE6+NO3

- it was more than that in RRE5+NO3- (Table

7). Figure 2. YEMA plate showing zone of clearance formed due to (A) phosphate solubilization and (B) pectin solubilization by B. cepacia (RRE5, RRE3 and RRE25), R. leguminosarum RRE6 and R. undicola RRE36

B

A B

5 (1): 8-14, May 2013 12

Figure 3. Effect of nitrate and R. leguminosarum RRE6 (A) and B. cepacia RRE5 (B) on seedling growth of maize. Note: 1-uninoculated control, 2-maize-RRE6 (A)/RRE5 (B), 3-maize-RRE6 (A)/RRE5 (B)+ NO3

-

Table 6. Effect of nitrate and bacterial inoculation on the growth of seedling of maize

Treatment Root length in cm plant-1

Shoot length in cm plant-1

Fresh weight in g plant-1

Control 4.76 2.7 0.56 RRE5strR 7.33* (53.99) 4.32* (60) 0.70* (25) RRE5strR+NO3

- 9.95* (109) 6.0* (122) 0.84* (50) RRE6strR 12.51* (162) 7.12* (163) 0.98* (75) RRE6strR+NO3

- 15.32* (221) 7.54* (64.1) 1.12* (100) CD at 1% 2.275 1.055 0.110 SEM ± 1.068 0.495 0.055

Note: RRE6strR = Streptomycin resistant mutant of Rhzobium; RRE5strR = Streptomycin resistant strain of Burkholderia; CD = Critical difference; SEM = Standard error of mean (n = 4). Values in parentheses indicate % increase over control. *significant at 1%

Table 7. Cell density recovered from root macerates of inoculated plants of maize

Treatment Cell density* (X 106cells ml-1)

RRE5strR 2.35±0.09 RRE5strR + NO3

- 5.72±1.02

RRE6strR 3.39±0.65 RRE6strR + NO3

- 6.79±1.35 Note: *Values are expressed as g-1 root fresh weight ml-1

Discussion Main objective of this study was to demonstrate the

effect of bacterial strain on maize genotype that provide increased plant productivity compared with the uninoculated control under fully sterilized conditions. Representatives of α subclass of Proteobacteria (R. undicola RRE36 and R. leguminosarum RRE6) and β subclass of Proteobacteria B. cepacia (RRE5, RRE3 and RRE25) were used for the plant growth promotion experiment. Results clearly indicated a significant increase in various plant growth parameters in presence of the above strains when compared to the control or uninoculated plant.

The most important parameter studied in this experiment was dry weight. When dry weight was considered both shoot and root dry weight were increased significantly when inoculated with the different endophytic strains. A similar association was described between maize and Rhizobium etli (Gutierrez-Zamora and Martinez-Romero 2001). The later authors reported an increase in maize plant dry matter upon R. etli inoculation. PGPR strains Pseudomonas and Bacillus significantly affected the height and dry weight of maize plants as was found by Jarak et al. (2012).

PGPR can enhance the growth and development of associated crops by improving nutrient uptake (Biswas et al. 2000). Shoot growth increased several folds in the present experiment..The exact mechanism of plant growth promotion by these isolates is not well understood in the case of maize. Phytohormone production, phosphate solubilization, nitrogen fixation and certain phenotypic changes like root proliferation could be the possible ways through which the host plants were benefited. PGPR uses one or more mechanisms to improve the growth and health of plants and can act simultaneously or independently at different stages of plant growth. Among these, phosphosolubilization, nitrogen uptake, and phytohormone production (indole-3-acetic acid), pectin solubilization was found to be present in all the strains. Large proportion of phosphorus in soil is insoluble and therefore unavailable to plants and hence phosphate solubilization is a desired property to be present in the bacteria. All tested rhizobial endophytes were able to solubilize phosphates and act as PGPR. They also act as biological agents through the production of siderophores.

Rhizobium-cereal associations are quite dynamic, enhancing the plant’s root architecture as well as the overall growth physiology. This finding suggests that endophytes get intimately associated with roots of maize seedling in a very early stage of development of plant. Nitrate uptake and root architecture are affected by PGPR and nitrate availability. Nitrate transporter genes are induced by the presence of external nitrate which elicits root elongation and biomass. The effects of PGPR on

1 2 3 1 2 3

A B


nitrate uptake are similar to those of low nitrate availability (Mantellin et al. 2003). Changes in root architecture similar to those induced by PGPR are due to the changes in nitrate availability in the medium (Wiersum 1958). In the present study, it was found that in the presence of nitrate (10mM) along with the strains, the growth of maize seedling was enhanced. It was found to be the highest in the case of R. leguminosarum RRE6, supplemented with nitrate. Alami et al. (1999) found that Rhizobium could be used in association with non-legume crops to better utilize the nutrients. The ability to utilize various nitrogenous compounds by rhizobial endophytes for their growth may be correlated with their evolutionary history. Due to extensive use of nitrogenous fertilizer in field, only those microorganisms could survive who had an inherent capacity to utilize the diverse nitrogenous compound. In the present experiment, it has been found that rhizobial inoculation to maize cultivar Malaviya 1 resulted in proliferated root architecture.

When nitrate was added as the supplement, growth was found to be more significant in all the cases (Table 6). Endophytic rhizobia may alter the morphological and physiological development of maize plant in ways that make them better miners of the existing resources of plant nutrients in soil. This has been reported in previous studies showing significantly increased production of root biomass in plants inoculated with certain rhizobial endophytes (Yanni et al. 1997; Praytino et al. 1999; Biswas et al. 2000; Yanni et al. 2001). It was found that maize seedling roots become intimately associated with bacterial cells in 48 h. During this initial period it was found that there was significant increase in all the growth parameters studied and endophytes recovered from the surface-sterilized roots in case of R. leguminosarum RRE6+NO3

- were found to be 6.79 x 106, which is higher than B. cepacia RRE5+NO3

- (5.72 x 106). Earlier, it was always thought that rhizobia did not enter non leguminous root tissues in any substantial manner (Sprent 1989). In the present study it was found that bacterial population obtained from nitrate enriched root macerates of maize was very high in both the strains. So it might be possible that these endophytes are entering inside the roots of maize seedling.

CONCLUSION

Both the bacterial groups, i.e., Burkholderia cepacia RRE5, RRE3 and RRE25 (member of β-subclass) and Rhizobium leguminosarum bv. phaseoli RRE6 and R. undicola RRE36 (member of α-subclass) were capable of establishing as endophytes of maize plant. In the presence of nitrate, the plant growth promotion effect produced by endophytes (RRE6 and RRE5) was more enhanced; RRE6 showed better effect as compared to RRE5. From this study it can be suggested that one of the sites of infection by endophytic bacteria is root of the host plant. The endophytic behavior and colonization ability of bacterial endophytes (RRE5 and RRE6) in the maize plants can be enhanced by nitrate supplementation.

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Effect of nitrogen fertilization on morphological and biochemical traits of some Apiaceae crops under arid region conditions in Egypt

KHALID ALI KHALID

Department of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. Tel. +202-3366-9948, +202-33669955, Fax: +202-3337-0931, ♥e-mail: [email protected]

Manuscript received: 14 January 2013. Revision accepted: 28 March 2013.

Abstract. Khalid KA. 2013. Effect of nitrogen fertilization on morphological and biochemical traits of some Apiaceae crops under arid region conditions in Egypt. Nusantara Bioscience 5: 15-21. Arid regions in Egypt are characterized by poor nutrients content and unfavorable environmental conditions which negatively affect growth and productivity of medicinal and aromatic plants including anise, coriander and sweet fennel. So the main objective of the present investigation was to study the effect of different levels of N, namely 0 (control), 100, 150 and 200 kg ha-1 used as ammonium sulphate [(NH4)2SO4] (20% N), on selected morphological and biochemical characteristics of anise, coriander and sweet fennel plants cultivated under arid regions conditions during two successive seasons. The most effective dose of nitrogen was 200 kg ha-1 of N, resulting in a positive increase in vegetative growth characters and content of essential oil, fixed oil, total carbohydrates, soluble sugars, protein and nutrients (NPK).

Key words: Anise, coriander, sweet fennel, essential and fixed oil, carbohydrates, protein

Abstrak. Khalid KA. 2013. Pengaruh pemupukan nitrogen pada sifat morfologi dan biokimia dari beberapa tanaman Apiaceae di kondisi daerah kering di Mesir. Bioscience Nusantara 5: 15-21. Daerah kering di Mesir yang ditandai dengan kandungan hara yang buruk dan kondisi lingkungan yang tidak menguntungkan yang berpengaruh negatif terhadap pertumbuhan dan produktivitas tanaman obat dan aromatik termasuk adas, ketumbar, dan adas manis. Tujuan utama penelitian ini adalah mempelajari pengaruh berbagai tingkat nitrogen, yaitu 0 (kontrol), 100, 150 dan 200 kg ha-1 yang digunakan sebagai sumber amonium sulfat [(NH4) 2SO4] (20% N), pada karakteristik morfologi dan biokimia tertentu dari tanaman adas, ketumbar dan adas manis yang dibudidayakan di bawah kondisi daerah kering selama dua musim berturut-turut. Dosis nitrogen yang paling efektif adalah 200 kg ha-1, mengakibatkan peningkatan positif dalam karakter pertumbuhan vegetatif serta kangdungan minyak atsiri, minyak tetap, karbohidrat total, gula larut, protein dan nutrisi (NPK).

Kata kunci: Adas, ketumbar, adas manis, minyak atsiri, minyak tetap, karbohidrat, protein

INTRODUCTION

Plant nutrition is one of the most important factors that increase plant productivity. Nitrogen (N) is the most recognized in plants for its presence in the structure of the protein molecule. In addition, N is found in such important molecules as purines, pyrimidines, porphyrines, and coenzymes. Purines and pyrimidines are found in the nucleic acids RNA and DNA, which are essential for protein synthesis. The porphyrin structure is found in such metabolically important compounds as the chlorophyll pigments and the cytochromes, which are essential in photosynthesis and respiration. Coenzymes are essential to the function of many enzymes. Accordingly, nitrogen plays an important role in the synthesis of the plant constituents through the action of different enzymes (Jones et al. 1991). Nitrogen limiting conditions increase volatile oil production in annual herbs. Nitrogen fertilization has been reported to reduce essential oil content in creeping juniper (Juniperus horizontalis) (Robert 1986), although it has been reported to increase total essential oil yield in thyme

(Thymus vulgaris L.) (Baranauskienne et al. 2003). Munsi 1992 indicated that for improvement in production of essential oil from a crop like Japanese mint (Mentha arvensis L), a judicious application of nitrogen is required. Each increase in N level increased the dill seed (Anethum graveolens L.) up to 90 kg ha-1 but further increase did not affect the seed yield significantly (Randhawa et al. 1996). Nitrogen fertilization increased the vegetative growth, essential oil, fixed oil, total carbohydrates, soluble sugars and NPK content of some Apiaceae (Anis, coriander and sweet fennel) and Nigella sativa L. plants (Khalid 1996; Khalid 2001). Zheljazkov and Margina (1996) established that plant height, branching, and essential oil content of mint (Mentha piperita and Mentha arvensis) increased with increasing N fertilizer rates. However, plant vegetative growth was not significantly affected by the increase of N fertilizer rates. With the increased N fertilizer rates, the fresh herbage yield from the first cut increased by 13 to 72%, and that from the second cut by 23 to 78% compared to the control. Nitrogen fertilization has been shown not only to improve vegetative growth, but also to alter the

5 (1): 15-21, May 2013 16

Figure 1. A. Anise (Pimpinella anisum L), B. Coriander (Coriandrum sativum L.), C. Sweet fennel (Foeniculum vulgare Mill.) (Photo: from many sources) essential oil yield and composition of Japanese mint (Mentha arvensis) (Saxena and Singh 1998). The influence of N fertilizers on the yield of crop, as well as on the production and composition of the essential oil and some other chemical characteristics of thyme was investigated by Baranauskienė et al. 2003. It was found that N fertilizer increased thyme crop, but differences in the yield of essential oil were not remarkable. However, the use of certain amounts of nitrogen fertilizers resulted in higher yields of essential oil obtainable from the cultivation area unit (dm3 ha-1). The effects of N fertilizer on yield and quality of basil (Ocimum basilicum L.) were investigated by Arabaci and Bayram (2004) and they found that intensive N fertilization increased the amount of green herb yield, essential oil concentration and essential oil yield. According to Ashraf et al. (2006), a field experiment was conducted to study the effect of N fertilization level on the content and composition of oil, essential oil and minerals in black cumin (Nigella sativa L.) seeds. Sixty-three-day-old plants were supplied with varying levels of N, i.e., 0, 30, 60, and 90 kg N ha−1. The fixed oil content of the seeds ranged from 32.7% to 37.8% and it remained almost unchanged at the two higher external N regimes, i.e., 60 and 90 kg N ha−1, but at 30 kg N ha−1 the oil content increased significantly; Increasing N rate did not affect the content of nutrient content in the cumin seeds. Akbarinia et al. (2007) indicated that with increasing of N to 60 kg ha-1, there was a significant increase in coriander (Coriandrum sativum L.) seed yield, but the highest essential oil and fatty acids content were obtained with 90 kg N ha-1. Senthil Kumar et al. (2009) revealed that application of N at 93.75 kg ha-1 gave the highest plant height, number of laterals, fresh and dry weight of shoot, dry matter production, fresh herbage yield and essential oil yield of Davana (Artemisia pallens Wall.). Nitrogen fertilization had different effects on mint chemotypes, with M. x piperita, linalool chemotype, being the only genetic material where nitrogen fertilization resulted in higher total dry mass (Luciana et al. 2010). Hellal et al. (2011) indicated that applying N fertilizer increased the growth, yield and chemical constituents of dill (Anethum graveolens L.) plant compared to the untreated control, the highest values of

vegetative growth, oil yield and NPK content were recorded by the treatment of 100 kg N ha -1.

Anise (Pimpinella anisum L., Apiaceae) has been used as an aromatic herb and spice since Egyptian times and antiquity and has been cultivated throughout Europe (Hänsel et al. 1999). In folk medicine, anise is used as an appetizer, tranquillizer and diuretic drug (Tyler et al. 1988; Lawless 1999). The traditional use of Pernod, Ouzo, Anisette, Raki, and many other anise-flavoured drinks after a heavy meal is a familiar example of its antispasmodic effect, especially in the digestive tract (Hänsel et al. 1999). Dried ripe fruits of anise, commercially called aniseeds (Anisi fructus), contain the whole dry cremocarp of anise (P. anisum). For medical purposes, they are used to treat dyspeptic complaints and catarrh of the respiratory tract, and as mild expectorants. It was also reported that extracts from anise fruits have therapeutic effects on several conditions, such as gynaecological and neurological disorders (Czygan and Anis 1992; Lawless 1999). Ethanolic extract of anise-fruits contains trans-anethole, methylchavicol (estragole), eugenol, psedoisoeugenol, anisaldehyde, coumarins (umbelliferon, scopoletin), caffeic acid derivatives (chlorogenic acid), flavonoids, fatty oil, proteins, minerals, polyenes and polyacetylenes as its major compounds (Hänsel et al. 1999).

Coriander (Coriandrum sativum L.) is a culinary and medicinal plant and belongs to the Apiaceae family. This plant has economic importance since it has been used as flavoring agent in food products, perfumes and cosmetics. As a medicinal plant, C. sativum L. has been credited with a long list of medicinal uses. Powdered seeds or dry extract, tea, tincture, decoction or infusion have been recommended for dyspeptic complaints, loss of appetite, convulsion, insomnia and anxiety (Emamghoreishi et al. 2001). Moreover, the essential oils and various extracts from coriander have been shown to possess antibacterial (Burt 2004), antioxidant (Wangensteen et al. 2004), anticancerous and antimutagenic (Chithra and Leelamma 2004) activities. Many phytochemical studies so far investigated the chemical composition of the essential oil from C. sativum L. fruits from different origins (Steinegger and Hänsel 1988). Evaluations of the essential oil

A B C

KHALID et al. – Biosensor for determination of ethanol in acetic acid 17

composition extracted from leaves have also been reported (Eyres et al. 2005). The coriander (Coriandrum sativum L.) fruit essential oil yields showed marked increase during maturation process and linalool was the main compound at the fruiting stage (Kamel et al. 1994).

Due to their unique and preferred flavor and aroma, the swollen bases of sweet fennel (Foeniculum vulgare var. dulce, Apiaceae) are freshly consumed in salads or cooked as a kitchen vegetable. The major constituents of fennel essential oil such as anethole and limonene are also used as essence in cosmetics and perfumes and for some medicinal purposes (Marotti et al. 1993; Stuart 1982).

Sandy soils generally have fine grained texture. They retain very little water, fertilizers or nutrients which means they are extremely poor. They are prone to over-draining and summer dehydration, and in wet weather can have problems retaining moisture and nutrients and can only be revitalized by the addition of organic matter. Sandy soils are light and easy to dig, hoe and weed. In addition, arid regions in Egypt are characterized by low nutrient contents (especially N) which negatively affect growth and productivity of medicinal and aromatic plants including anise, coriander and sweet fennel (Abd-Allah et al. 2001). The main objective of the present investigation was to study the effect of different levels of N fertilizers on the morphological and biochemical contents of anise, coriander and sweet fennel plants under arid regions conditions.


Experiments were carried out in the arid region at the Experimental Farm of the Desert Development Center (DDC) in Sadat City, American University, Egypt, during two successive seasons, 1992/93 and /1993/94. The area of DDC had been recently reclaimed and had not been cultivated before. Physical and chemical properties of the Typic Torrifluvents soil (USDA 1999) used in this study (0-50 cm depth) were determined according to Jackson (1973) and Cottenie et al. (1982) and are presented in Table 1. Seeds of coriander and anise were provided by the Department of Medicinal and Aromatic Plants, Ministry of Agriculture, Giza, Egypt as follows: fertilizers were added to all plots as follows: cattle manure (50 m3 ha-1), phosphorus (500 kg ha-1) as calcium super phosphate (15.5% P2O5) and potassium (375 kg ha-1) as potassium sulphate (48% K2O); whereas sweet fennel seeds were imported from France. Sweet fennel seeds were sown in the third week of October during both seasons. The seedlings of sweet fennel were transplanted into the open field 45 days after sowing. At the same time, the seeds of coriander and anise were sown directly in the open field. The experimental design was a complete randomized block with four replicates. The experimental area (plot) was 30 m² (4 m x 7.5m) containing 15 rows; the distance between hills was 25 cm and 50 cm between the rows. Thinning for two plants per hill was made 45 days after cultivating the plants in the open field. The sprinkler irrigation system was used in this experiment. All agriculture practices other than experimental treatments were performed according to the

recommendations of the Ministry of Agriculture, Egypt. Plots were divided into four groups subjected to N application to soil as ammonium sulphate [(NH4)2SO4] (20% N) with the rates of 0, 100, 150 and 200 kg ha-1. Table 1. Mechanical and chemical analyses of the Typic Torrifluvents soil

Sand Silt Clay Gravel pH ECe (dS m-1) %

79.7 13.0 7.3 18.7 8.7 2.0

Ca++ Mg++ Na++ K+ CO3 HCO3- Cl - SO4

-- mg g -1

4.9 5.6 1.9 0.6 0.8 1.9 18.6 0.2

K Fe Cu Zn Mn mg g -1 ug mg-1

0.5 5400 400 300 1600

Harvesting At fruiting stage, the plants were harvested during the

two seasons. Vegetative growth characters measurements [Plant height (cm), Leaf number (plant-1), Branch number (plant-1), Umbel number (plant-1), Herb fresh weight (g plant-1), Herb dry weight (g plant-1) and Fruit yield (g plant-

1)] were recorded.

Essential oil isolation Ripening fruits were collected from each treatment

during the first and second season, and then 100 g from each replicate of all treatments was subjected to hydro-distillation for 3 h using a Clevenger type apparatus (Clevenger 1928). The essential oil content was calculated as a weight/volume percentage. In addition, total essential oil yield (g plant-1) was calculated by using the dry weight of the fruits.

Total carbohydrates and soluble sugars Total carbohydrates (TC) and soluble sugars

concentrations in leaves (collected at the end of the first and second season of each treatment) were determined according to Ciha and Brun (1978) with some modifications. Samples of 100 mg were homogenized with 10 mL of extracting solution [glacial acetic acid: methanol: water, 1:4:5, v/v/v for soluble sugars or glacial acetic acid: H2SO4 (1N): water, 1:4:5, v/v/v for TC]. The homogenate was centrifuged for 10 min at 3,000 rpm and the supernatant was decanted. The residue was resuspended in 10 mL of extracting solution and centrifuged another 5 min at 3,000 rpm. The supernatant was decanted, combined with the original extract and made up to 50 mL with water. For measurement of total carbohydrates and soluble sugars, a phenol-sulfuric acid assay was used (Dubois et al. 1956). A volume of 0.5 mL of 5% (v/v) phenol solution and 2.5 mL of concentrated sulfuric acid were added to 0.5 mL aliquots. The mixture was shaken, heated in a boiling water-bath for 20 min and cooled to room temperature. The absorption was then determined by spectrophotometry at 490 nm.

5 (1): 15-21, May 2013

18

Fixed oil, nutrients and crude protein determination Fixed oil Extraction: Fifty grams of fruits were crushed

to coarse powder and extracted with petroleum ether (40-60 ο C) in a Soxhlet apparatus (AOAC 1970). N, protein, P and K (in the leaves) of both seasons of each treatment were determined using the methods described by the AOAC (1970) as follows:

The washed and dried materials were ground to fine powder with mortar and pestle and used for dry ashing.

For analysis of K, the powdered plant material (0.2 g) was taken in precleaned and constantly weighed silica crucible and heated in muffle furnace at 400 0C till there was no evolution of smoke. The crucible was cooled in desicator at room temperature. The ash, totally free from carbon was moistened with conc. H2SO4 and heated on hot plate till fumes of sulphuric acid was evolved from the silica crucible, with sulphated ash was again heated at 600 0C in muffle furnace till weight of sample was constant (3-4 hrs). One gram sulphated ash was dissolved in 100 ml 5 % conc. HCl in a beaker to obtain solution for determination of K through flame photometry. Standard solution of each mineral was prepared and calibration curve drawn for K element using flame photometry (Alexander 1963).

For the determination of protein and nitrogen using Micro Kjeldahl method, 1 g of plant sample taken in a Pyrex digestion tube and 30 ml of conc. H2SO4 carefully added, then 10 g potassium sulphate and 14 gm of copper sulphate, mixture was placed on sand both on a low flame just to boil the solution. It was further heated till the solution became colorless and clear, allowed to cool, diluted with distilled water and transferred to 800 ml Kjeldahl flask, washing the digestion flask, three or four pieces of granulated zinc and 100 ml of 40 % caustic soda were added and the flask was connected with the splash heads of the distillation apparatus. Next 25 ml of 0.1 N sulphuric acids was taken in the receiving flask and distilled; it was tested for completion of reaction. The flask was removed and titrated against 0.1 N caustic soda solution using Methyl Red indicator for determination of nitrogen, which can be calculated to give the protein content (Lynch and Barbano 1999).

For determination of phosphorous, a 2 g sample of plant material was brought into a 100 ml conical flask, two spoons of Darco-G-60 were added followed by 50 ml of 0.5 M NaHCO3 solution. Next, the flask was corked and shaked for 30 min on a shaker. The content was filtered and the filtrate was collected in a flask from which 5 ml filtrate were taken to a 25 ml volumetric flask. To this solution 2 drops of 2, 4- paranitrophenol and 5 N H2SO4 drops were added with intermittent shaking till the yellow color disappeared. The content was diluted to 20 ml with distilled water and then 4 ml ascorbic acid were added. Then the mixture was well shaken and the intensity of blue color at 660 nm on colorimeter was measured and compared to phosphorus standards to obtain P concentrations (King 1932).

Statistical analysis In this experiment, one unique factor was considered:

nitrogen application as ammonium sulphate (0, 100, 150

and 200 kg ha-1 ). For each treatment there were four replicates. The experimental design followed a complete random block design. The averages of data for both seasons were statistically analyzed using one way analysis of variance (ANOVA-1) using the STAT-ITCF program (Foucart 1982). The least of significant differences between means was calculated using least significant difference (LSD) at 5% according to Snedecor and Cochran (1990).


Effect of N fertilization on plant growth and development

Data in Table 2 shows the response of anise, coriander and sweet fennel plants to the different rates of N. All Nitrogen treatments produced significantly higher values than the control and significantly improved plant growth characters [plant height (cm), leaf number (plant-1), branch number (plant-1), umbel number (plant-1), herb fresh weight (g plant-1), herb dry weight (g plant-1) and fruit yield (g plant-1)]. Their highest values were recorded when plants were treated with 200 kg N ha-1. The highest values of plant growth characters (respectively) were 44.5, 32.6, 8.5, 29.7, 19.8, 9.4 and 5.9 for anise; 80.0, 66.0, 7.1, 29.9, 15.9, 12.3 and 5.9, for coriander; 98.9, 21.3, 5.3, 10.4, 118.4, 90.4 and 21.3, for sweet fennel. The ANOVA indicated that the increases in vegetative growth characters were significant for N treatments (Table 2). The positive effects of N fertilization may be due to the important physiological role of N in molecule structure as porphyrin. The porphyrin structure is found in such metabolically important compounds as the chlorophyll pigments and the cytochromes, which are essential in photosynthesis and respiration. Coenzymes are essential to the function of many enzymes. Accordingly, nitrogen plays an important role in synthesis of the plant constituents through the action of different enzymes activities and protein synthesis (Jones et al. 1991) that reflected in the increase in growth parameters of plants such as anise, coriander and sweet fennel plants. Also, these results are in accordance with those obtained by Khalid (1996, 2001) on some Apiaceae and Nigella sativa L. plants; Ashraf et al. (2006) on cumin; Akbarinia et al. (2007) on coriander; Hellal et al. (2011) on dill (Anethum graveolens L.), all of whom reported that N fertilizer treatments were superior to the control treatment and significantly improved the vegetative growth characters of family Apiaceae.

Effect of N fertilization on the essential oil content The effects of different treatments of N on the essential

oil content (% or ml plant-1) extracted from anise, coriander and sweet fennel fruits are represented in Table 3. Generally, all levels of N as soil application progressively increased the essential oil of anise, coriander and sweet fennel plants compared to the control. The last level of N (200 kg N ha-1) seemed to be optimal for obtaining a higher concentration of essential oil than the control and other treatments: 0.6, 0.1 and 0.7% more than the control for anise, coriander and sweet fennel, respectively. The


ANOVA indicated that the increase in essential oil (%) was significant (P<0.05) in anise and not significant in coriander and sweet fennel. The increase in essential oil yield (g plant −1) were not significant in anise and coriander but was significant in sweet fennel (Table 3). The effect of different N treatments on essential oil may be due to its effect on enzyme activity and metabolism of essential oil production in peppermint plant (Burbott and Loomis 1969). These results were in accordance with those obtained by Khalid (1996 and 2001) on some Apiaceae and Nigella sativa L. plants; Ashraf et al. (2006) on cumin; Akbarinia et al. (2007) on coriander; Hellal et al. (2011) on dill (Anethum graveolens L.), who reported that N has a positive effect on the quantity of essential oil extracted from Apiaceae plants.

Effect of N fertilization on the fixed oil content

Data presented in Table 3 shows that the intensive N fertilization produced an increase in the accumulation of fixed oil (% or ml plant-1) extracted from anise, coriander and sweet fennel fruits, with the highest content of fixed oil obtained with the highest N dose of 200 kg ha-1. Fixed oil contents were 8.1, 2.7 and 0.4% higher than the control for anise, coriander and sweet fennel, respectively. The ANOVA indicated that the increase in fixed oil (%) of anise, coriander and sweet fennel was significant. The increase in fixed oil yield (ml plant-1) was significant for anise and coriander while not significant for coriander. These results were similar to those of Khalid (1996) on some Apiacea plants; Khalid (2001) a Ashraf et al. (2006) on Nigella sativa L.; and Akbarinia et al. (2007) on coriander (Coriandrum sativum L.)

Effect of N fertilization on the total carbohydrates and soluble sugars contents

Table 3 shows that total content of carbohydrates and soluble sugars in anise, coriander and sweet fennel increased with increasing N rates. The highest contents of total carbohydrates and soluble sugars were recorded when plants were treated with 200 kg ha-1 compared with the control or other treatments. Total carbohydrates were 0.6%, 7.1% and 0.4% higher than the control and total soluble sugars, 0.8%, 3.2%, and 0.4%) for anise, coriander and sweet fennel, respectively. The ANOVA indicated that the increase in total carbohydrates in anise and coriander was significant, while it was not significant for sweet fennel.

The increase in soluble sugars in anise and sweet fennel

was not significant, but it was significant in coriander. These results may be due to the increase in chlorophyll content, and consequently, photosynthesis efficiency, induced by N. So it showed that total carbohydrates and

Table 2. Effect of N fertilization on growth characters (at fruiting stage) and fruit yield

Nitrogen treatments

(kg ha-1)

Growth characters

Plant height (cm)

Leaf number (plant -1)

Branch number (plant -1)

Umbel number (plant -1)

Plant fresh

weight (plant-1)

Plant dry weight

(plant -1)

Fruit yield (g plant -1)

Anise (Pimpinella anisum) 0 31.0 15.7 4.6 12.0 5.2 2.9 1.1 100 40.7 31.5 7.5 24.0 12.4 6.4 3.9 150 42.3 31.6 7.7 25.6 19.7 9.0 5.8 200 44.5 32.6 8.5 29.7 19.8 9.4 5.9 LSD: 0.05 2.1 8.3 0.6 3.9 5.8 2.4 0.8 Coriander (Coriandrum sativum) 0 41.2 14.2 2,8 7.5 4.3 2.8 1.2 100 68.8 46.1 6.5 29.3 13.0 8.7 3.1 150 69.3 63.8 6.7 29.7 13.9 8.8 3.8 200 80.0 66.0 7.1 29.9 15.9 12.3 5.9 LSD: 0.05 7.5 10.7 0.9 3.6 2.9 2.4 0.9 Sweet fennel (Foeniculum vulgare var. dulce) 0 62.4 8.8 1.5 1.4 66.8 44.6 10.6 100 83.8 16.9 4.3 10.1 106.7 77.7 19.7 150 95.2 19.4 4.6 10.2 114.1 77.9 19.9 200 98.9 21.3 5.3 10.4 118.4 90.4 21.3 LSD: 0.05 6.2 4.2 0.7 1.3 18.5 10.1 3.2 Table 3. Effect of N fertilization on chemical composition

Nitrogen treatments

(kg ha-1)

Chemical constituents Essential oil Fixed oil Total

car. (%)

Soluble sugars

(%)

Protein (%)

N (%)

P (%)

K (%)(%) ml

plant-1(%) ml

plant-1

Anise (Pimpinella anisum) 0 2.6 0.11 7.0 0.1 23.7 2.7 14.4 2.3 0.7 1.5 100 2.7 0.14 9.0 0.2 28.8 3.0 15.5 2.6 0.8 1.6 150 2.8 0.18 10.7 0.2 24.1 3.3 16.6 2.7 0.9 1.9 200 3.2 0.21 15.1 0.2 24.3 3.5 16.8 2.9 1.0 2.0 LSD: 0.05 0.1 NS 1.5 NS 2.6 NS 0.2 0.3 NS NS Coriander (Coriandrum sativum) 0 0.2 0.01 5.5 0.1 15.3 1.9 9.4 1.6 0.3 2.9 100 0.2 0.02 6.9 0.2 17.0 4.3 9.7 1.8 0.4 3.1 150 0.2 0.02 7.5 0.3 17.3 4.4 11.8 1.9 0.5 3.2 200 0.3 0.03 8.2 0.5 22.4 5.1 14.3 2.8 0.5 3.3 LSD: 0.05 NS NS 1.2 0.1 1.0 0.6 0.9 0.1 NS NS Sweet fennel (Foeniculum vulgare var. dulce) 0 1.3 0.11 1.2 0.1 18.4 1.7 12.9 1.8 0.6 2.1 100 1.9 0.41 1.3 0.3 18.5 1.8 13.0 1.9 0.7 2.3 150 2.0 0.41 1.6 0.3 18.6 2.0 13.3 2.0 0.8 2.5 200 2.0 0.42 1.6 0.4 18.8 2.1 13.3 2.1 0.9 2.6 LSD: 0.05 NS 0.11 0.1 0.1 NS NS NS NS NS 0.1

5 (1): 15-21, May 2013

20

soluble sugars contents increased with application of N (Jones et al. 1991).

Effect of N fertilization on the total protein content Protein content was positively affected by soil

application of N (Table 3). The treatment of 200 kg ha-1

resulted in the highest protein content in anise, coriander and sweet fennel plants: 2.4%, 3.9% and 0.4% higher than the control, respectively. The ANOVA indicated that the increase in total protein content was significant for anise and coriander while not significant for sweet fennel. These results may be due to the influence of N on the ribosome structure and the biosynthesis of some hormones (gibberellines, auxins, cytokinins) involved in protein synthesis (Jones et al. 1991; El-Wahab and Mohamed 2007).

Effect of N fertilization on the mineral content It is evident from the Table 3 that NPK content

gradually increased in all treatments as compared with the control treatment. With respect to the effect of the N levels, data indicated that applying 200 kg ha-1 brought about the highest values of NPK content in anise, coriander and sweet fennel plants: 0.6%, 0.3% and 0.5% for anise; 1.2%, 0.2 % and 0.4% for coriander; 0.3%, 0.3%, and 0.5% for sweet fennel higher N, P and K than the control respectively. The ANOVA indicated that the increase in N content of anise and coriander was significant while it was not significant for sweet fennel. The increase in P content of anise, coriander and sweet fennel was not significant. The increase in K content of anise and coriander was not significant, but the increase in sweet fennel was. The increase in the essential minerals according to the N treatments may be due to the increase in the dry matter of plant materials (El-Wahab and Mohamed 2007).

CONCLUSION

It may be concluded that N treatments resulted in positive increase in plant growth characters, and content of essential oil, fixed oil, total carbohydrates, soluble sugars, protein, and nutrients. The highest values were recorded when plants were treated with 200 kg ha-1 for anise, coriander and sweet fennel.

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Response of Silybum marianum plant to irrigation intervals combined with fertilization

SABER F. HENDAWY1,♥, MOHAMED S. HUSSEIN1, ABD-ELGHANI A. YOUSSEF2, REYAD A. EL-MERGAWI3

1Medicinal and Aromatic Plants Research Department, National Research Centre, Dokki 12311, Giza, Egypt. Tel. +202-3366-9948, +202-33669955, Fax: +202-3337-0931, ♥email: [email protected]

2Departement of Chemistry, Faculty of Science, Jazan University, Saudi Arabia 3 Department of Plant Production and Protection, College of Agriculture and Veterinary Medicine, Qassim University,P.O.Box 6622, Buhrida 51452, Al-

Qassim; Saudi Arabia

Manuscript received: 11 April 2013. Revision accepted: 5 May 2013.

Abstract. Hendawy SF, Hussein MS, Youssef AA, El-Mergawi RA. 2013. Response of Silybum marianum plant to irrigation intervals combined with fertilization. Nusantara Bioscience 5: 22-29. This study was investigated to evaluate the influence of different kinds of organic and bio fertilization under different irrigation intervals on the growth, production and chemical constituents of Sylibium marianum plant. Data indicated that all studied growth and yield characters were significantly affected by the duration of irrigation intervals. Fertilizer treatments had a primitive effect on growth and yield characters. The interaction between irrigation intervals and fertilizer treatments has a clear considerable effect on growth and yield characters. The obtained results indicated the favorable effect of organic and bio fertilizers which reduce the harmful effect of water stress. Different treatments had a pronounced effect on silymarin content.

Key words: Sylibium marianum, silymarin, bio fertilization and irrigation intervals.

Abstrak. Hendawy SF, Hussein MS, Youssef AA, El-Mergawi RA. 2013. Respons tanaman Silybum marianum terhadap interval irigasi yang dikombinasi dengan pemupukan. Nusantara Bioscience 5: 22-29. Penelitian ini bertujuan untuk mengevaluasi pengaruh berbagai jenis pupuk organik dan hayati dengan interval irigasi yang berbeda terhadap pertumbuhan, produksi dan kandungan kimia tanaman Sylibium marianum. Data menunjukkan bahwa semua sifat pertumbuhan dan produksi yang diteliti secara signifikan dipengaruhi oleh durasi interval irigasi. Perlakuan pemupukan berpengaruh nyata terhadap karakter pertumbuhan dan hasil panen. Interaksi antara interval irigasi dan perlakuan pemupukan berpengaruh besar pada karakter pertumbuhan dan hasil panen. Hasil yang diperoleh menunjukkan efek menguntungkan dari pupuk organik dan hayati yang dapat mengurangi efek berbahaya cekaman air. Perlakuan yang berbeda berpengaruh kuat terhadap kandungan silymarin.

Kata kunci: Fagus orientalis, serat, sifat biometri, pohon unggul.

INTRODUCTION

Milk thistle (Silybum marianum L. Gaertn.), a member of the Mediterranean Basin, as a crop and weed on agricultural plantations, it occurs in many European countries, North Africa, South and North America, Central and Western Asia and southern Australia (Carrier et al. 2002).The pharmaceutical compound of milk thistle is derived from its fruits, which are achenes (Fructus silybi mariani). In their dry pericarp and seed coat the plant accumulates a group of flavonolignans commonly referred to as silymarin (Cappelletti and Caniato 1984). Taxifolin is their precursor. The main flavonolignans of milk thistle are silybinin, isosilybinin, silydianin and silychristin. Several other compounds of that type have also been identified, but their importance in the silymarin complex is insignificant (Kurkin et al. 2001). Silymarin, derived from the seeds of milk thistle plant has been used widely for the treatment of toxic liver damage (Dewick 1998). Silymarin primarily consists of an isomeric mixture of six phenolic compounds:

silydianin, silychristin, diastereoisomers of silybin (silybin A and B), and diastereoisomers of isosilybin (isosilybin A and B) (Lee et al. 2007).

The compost must be added to conventional NPK fertilizer to improve soil structure, making the soil easier to cultivate, encouraging root development, providing plant nutrients and enabling their increased uptake by plants. Moreover, compost aids water absorption and retention by the soil, reducing erosion and run-off and thereby protecting surface waters from sedimentation, help binding agricultural chemicals, keeping them out of water ways and protecting ground water from contamination (leaMaster et al. 1998). Compost has already been established as a recommended fertilizer for improving the productivity of several medicinal and aromatic plants, as peppermint (O’Brien and Barker 1996), Tagetes erecta (Khalil et al. 2002), Sideritis montana (El-Sherbeny et al. 2005), Ruta graveolens (Naguib et al. 2007) and Dracocephalum moldavica L. ( Amer 2008). Compost tea is a highly concentrated microbial solution produced by extracting

HENDAWY et al. – Response of Silybum marianum to irrigation and fertilizer 23

beneficial microbes from vermicompost and or compost. Compost tea provides direct nutrition as a source of foliar and soil organic nutrient and as chelated micronutrients for easy plant absorption. Also, compost tea provide microbial functions, that compete with disease causing microbes, degrade toxic pesticides, produce plant growth hormones, mineralize plant available nutrients and fix nitrogen (Hendawy 2008).

Arbuscular mycorrhiza (AM) fungi (Endogonaceae) form a mutualistic relationship with the roots of most plant species. This plant-fungus association involves the translocation of carbon from the plant to the fungus and enhanced uptake and transport of soil nutrients, primarily phosphorus, to the plant via the fungus (Newman and Reddel 1987). Other potential benefits of AM fungal colonization to host plants include improved uptake of poorly mobile nutrients such as zinc (Gildon and Tinker 1983), improved plant water relations (Allen and Allen 1986) and reduced pathogenic infections (Newsham, et al. 1995). AMF can also benefit plants by stimulating the production of growth regulating substances, increasing photosynthesis, improving osmotic adjustment under drought and salinity stresses and increasing resistance to pests and soil borne diseases (Al-Karaki 2006).

However, water deficit is a limiting factor in production of many field crops (Kafi and Mahdavi Damghani 2001; Munns 2002) and water stress causes different morphological, physiological and biochemical changes including: leaf area reduction, leaf senescence and reduction in cell development (Kafi and Mahdavi Damghani 2001), stomatal closure (Safar-Nezhad 2003) and photosynthetic limitation (Kafi and Mahdavi

Damghani 2001). It appears that the effect of water stress on economic yields of medicinal plants which are mainly secondary metabolites, are somehow positive (Baher et al. 2002). In many cases, a moderate stress could enhance the content of secondary metabolites.

This current experiment targeted the evaluation of the influence of different kinds of organic and bio fertilization under different irrigation intervals on the growth, production and chemical constituents of Sylibium marianum plant.


Field experiment Location

The field experiment was carried out at El-Nubareia Research Station (El-Behira Governorate, Egypt), National Research Centre, to investigate the influence of Chemical, organic and bio fertilizers under different irrigation intervals on growth, yield and chemical constituents of milk thistle.

Soil

The experiment was set up on sand loam soil as shown in Table 1.

Table 1. Main characteristics of soil Characteristics Value Mechanical analysis

Sand% 68.08 Silt% 16.00 Clay% 15.92 Texture Sandy loam

Chemical analysis PH 1:2.5ext. 8.50 Ca Co3 21.70

Electrical conductivity 1:2.5ext 0.61 Soluble cations meq/l

Ca++ 3.38 Mg++ 3.62 Na+ 3.23 K+ 0.49

Soluble anions meq/l HCO3

- 1.12 Cl- 1.5 SO4

- 9.1 Macro-elements (ppm)

N 30.00 P 20.00 K 368.00

Micro-elements (ppm) Zn 0.28 Mn 2.50 Fe 3.70 Cu 0.96

Figure 1. Inflorescense of milk thistle (Silybum marianum L. Gaertn.)

5 (1): 22-29, May 2013

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Experiment design and agronomic practices The fertilization factor experiment was set up in a

randomized design in three replicates.

Experimental treatments

A. Irrigation every 3 days 1. NPK (100 kg super phosphate+150 kg nitrate ammonium+50

kg potassium sulphate). 2. Compost 20m3/feddan 3. Compost 20m3/feddan+mycorrhiza 4. Compost 20m3/feddan+compost tea 20 L/feddan 5. Compost 20m3/feddan+compost tea 20 L/feddan+mycorrhiza B. Irrigation every 6 days 6. NPK (100 kg super phosphate+150 kg nitrate ammonium+50

kg potassium sulphate) as control. 7. Compost 20m3/feddan 8. Compost 20m3/feddan+mycorrhiza 9. Compost 20m3/feddan+compost tea 20 L/feddan 10. Compost 20m3/feddan+compost tea 20 L/feddan+mycorrhiza C. Irrigation every 9 days 11. NPK (100 kg super phosphate+150 kg nitrate ammonium+50

kg potassium sulphate) as control. 12. Compost 20m3/feddan 13. Compost 20m3/feddan+mycorrhiza 14. Compost 20m3/feddan+compost tea 20 L/feddan 15. Compost 20m3/feddan+compost tea 20 L/feddan+mycorrhiza

The seeds were directly sown in 20th of October 2010.

Each plot was 13.5 m2 consisting of 9 rows with a distance of 50 cm between the rows and 30 cm between each successive plant.. Weeding and thinning was done after 30 days of plantation. Recommended agronomic practices were adopted.

Super phosphate or compost was added during preparing soil. The other chemical fertilizers (Ammonium nitrate and Potassium sulphate were divided into two equal portions during the growing season, the 1st portion was added after one month of sowing, while the second one was applied after one month from the 1st. Tea compost (Table 2, 3) was sprayed after 60 days from sowing and repeated after 15 days.Vesicular arbscular mycorrhiza (VAM) fungi which contained 3 effective strains representing Glomus etunicatum, Glomus fasciculatum and Glomus intraradices. VAM fungi was used for soil inoculation. The VAM inoculation was applied into sowing hills at a rate of 5 mL/hill. The amount contained about 200 VAM spores/hill.

The effect of the above treatments was measured by plant height, branches number, capitula number/plant, seed yield and silymarin content.

Table 2. Microbial population of organic compost tea Constituent Value Bacterial Plate Count (CFU/ml) 7.1 X 107

Bacterial Direct Count (Cell/ml) 6.4 X 108

Spore Forming Bacteria (CFU/ml) 7 X 104

Total Fungi (CFU/ml) 2.8 X 105

Table 3. Chemical analysis of organic compost tea Constituent Value Bulk Density kg/m3 510 Moisture Content% 18.2 Electrical conductivity dS/m 9.65 PH 7.6 Total Organic Carbon% 24.6 Total Organic Matter% 42.41 Total Nitrogen% 1.35 C/N Ratio 18.22 NH4-N, mg/kg 880 NO3-N, mg/kg 450 Total Phosphorus% 1.6 av. Phosphorus mg/kg 410 Total Potassium% 2.3 av. Potassium mg/kg 620 Trace Element (ppm) Fe 960 Zn 280 Mn 320 Cu 140 Note: Nematodes (nil), Weeds germination (nil), Parasites (nil), Pathogenic (nil), Humus value (5)

Extraction procedure Silymarin content was extracted according to ( Cacho et

al. 1999). Gram of seeds were defated in a Soxhlet apparatus with 50 mL of petroleum-ether at 40-60 oC for 12 h. The residue was extracted with 50 mL of methanol at 65-70 oC over 8 h. The methanolic solution was concentrated to a dry residue. The extract was dissolved in 10 mL of methanol.

HPLC analysis HPLC was carried out using an HPLC pump monitored

at 280 nm by a UV detector and quantified by an integrator. A Shim-pack C18 ( 1250 x 4.6 mm ID) column was used, eluting with MeOH-H2O-AcOH 40:60:5, at a flow rate of 2 mL/min. Mixture of flavonolignans obtained from Alex Pharm, Egypt (specifications: Silychristin 25% Rt 2.94 min, silydianin 9.7% Rt 3.64 min, silybin A 21.3% Rt 7.84 min, silybin B 32% Rt 9.18 min, isosilbin A 8.7% Rt 13.61 min and isosilybin B 3% Rt 15.18 min).


Vegetative growth and yield Irrigation intervals

Data tabulated in Table 4 indicated that all studied growth and yield characters were significantly affected by the duration of irrigation intervals.

By increasing the severity and duration of drought from 3 days to 9 days, plant height (cm) showed significant reduction. Such reduction in plant height in response to drought may be due to blocking up of xylem and phloem vessels thus hindering any translocation through (Lovisolo and Schuber 1998). Similar results were obtained by Singh et al. (2006) and Khalil et al. (2010).


Table 4. Effect of irrigation intervals on vegetative growth and yield of Silybum marianum

Seed yield (g/Plant)

Flowers heads

no/plant

Branches no/plant

Plant height (cm)

Irrigation intervals

20.91 20.60 6.60 179.00 3 days17.39 18.20 7.20 169.40 6 days14.89 15.80 8.60 166.2 9 days 0.477 0.582 0.576 0.504 LSD at 5%

Data on hand, illustrated also that, number of

branches/plant increased significantly with decreasing of irrigation, this may be due to that drought reduced cycling-dependent kinase activity results in slower cell division as well as inhibition of growth (Schuppler et al. 1998). This supported by the results of (Rahmani et al. 2008) on Calendula officinalis L. and (Taheri et al. 2008) on Cichorium intybw L.

Significant higher numbers of flowers head/plant and seed yield (g/plant) were recorded with the shortest irrigation interval (3 days) followed by (6 days). The decrease in yield attributes under the longest irrigation interval (9 days) may be due that water stress changing the hormonal balance of mature leaves, thus enhancing leaf senescence and hence the number of active leaves decreased, as well as leaf area was reduced by water shortage, which was attributed to its effect on cell division and lamina expansion. When the number of active leaves decreased the light attraction and CO2 diffusion inside the leaf decreased and the total capacity of photosynthesis decreased, therefore, the photosynthetic materials that transferred to seeds will decreased (Ahmed and Mahmoud 2010; Moussavi et al. 2011).

Fertilizer treatments Data tabulated in Table 5 show that fertilizer

treatments had a significant effect on growth and yield characters of Silybum marianum plants. The mean values of plant height were 174.33, 164.33, 168.33, 171.0 and 179.67 cm as a result of NPK, compost, compost+ mycorrhiza, compost+compost tea and compost+compost tea+mycorrhiza treatments, respectively. So, the highest value of plant height was obtained as a result of compost+compost tea+mycorrhiza treatment.

Table 5. Effect of fertilizer treatment on vegetative growth and yield of Silybum marianum

Seed yield

(g/plant)

Flowers heads

no/plant

Branches No/plant

Plant height (cm)

Fertilizer treatments

18.15 21.33 8.33 174.33 NPK 15.74 17.00 6.33 164.33 Compost 18.90 16.67 7.33 168.33 Compost+mycorrhiza16.49 17.00 7.33 171 Compost+compost tea19.37 19 8.00 179.67 Compost+compost

tea+mycorrhiza1.054 0.318 0.449 0.825 LSD at 5%

The results in Table 5 reveal that, fertilizer treatments had a pronounced effect on branches number. It can be noticed that, mean values of branches number recorded 8.33, 6.33, 7.33, 7.33 and 8.00/plant were obtained from NPK, Compost, Compost+mycorrhiza, compost+compost tea and compost+compost tea+mycorrhiza treatments, respectively. Thus, the maximum mean value of branches number/plant (8.33) was obtained as a result of NPK treatment followed by compost+compost tea+mycorrhiza treatment, which recorded 8.00/plant. There is no significant difference between NPK treatment and compost+compost tea+mycorrhiza treatment.

The averages of heads flowers number were 21.33, 17.00, 16.67, 17.00 and 19.00/plant as a result of NPK, Compost, Compost+mycorrhiza, compost+compost tea and compost+compost tea+mycorrhiza treatments, respectively. Thus, the maximum mean value of flowers heads number/plant (21.33) was obtained from NPK treatment followed by compost+compost tea treatment, which recorded 19.00/plant.

It is evident from data in Table 5 that fertilizer treatments had a significant effect on seed yield (g/plant).In this respect, mean values of seed yield (g/plant) were 18.15, 15.74, 18.90, 16.49 and 19.37 g/plant as a result of as a result of NPK, compost, compost+mycorrhiza, compost+compost tea and compost+compost tea+ mycorrhiza treatments, respectively. Therefore, compost+ compost tea+mycorrhiza treatment gave the highest mean value of seed yield (19.37g/plant) followed by compost+mycorrhiza treatment which recorded (18.90 g/plant).

The promotion effect of compost on the growth and yield of plant could be explained through the role of organic materials including composts in improving soil P availability (Gichangi et al. 2009). Since during composting, labile nutrients are converted into stabilized organic material (Zucconi and De Bertoldi 1987), therefore a large proportion of nutrients are labile. Composts provide microbes not only with P but also C and N and are therefore likely to induce changes in P pools that differ from those of inorganic P addition (Hassan et al. 2012).

The favorable effects of the combination between compost +compost tea+mycrohiza may be explained based on the beneficial effects of them on the improvement soil physical and biological properties and also, the chemical characteristics resulting in more release of available nutrient elements to be absorbed by plant root and its effect on the physiological processes such as photosynthesis activity as well as the utilization of carbohydrates. A similar suggestion was made by Hanafy et al. (2002) on rocket plants. Furthermore, this stimulative effect may be related to the good equilibrium of nutrients and water in the root medium (Abdelaziz and Balbaa 2007) or to the beneficial effects of mycorrhiza on vital enzymes and hormonal, stimulating effects on plant growth and yield.

Interaction treatments The interaction between irrigation intervals and

fertilizer treatments has a clear considerable effect on growth and yield characters (Table 6). It can be observed

5 (1): 22-29, May 2013

26

that the maximum mean value of plant height (190.00 cm) was obtained from the combination treatment between irrigation intervals every 3 days and fertilized with compost+compost tea+mycorrhiza. On the other hand, the lowest average of plant height (158.00 cm) was obtained from the combination between irrigation intervals every 9 days and compost treatment. The variation in plant height between maximum and the minimum values reached to 20.25%.

For branches number/plant, it can be observed that, the highest mean value of branches number/plant (10.00/plant) against the lowest value (5.00/plant) were obtained as a result of the combination between irrigation intervals every 9 days and NPK treatment and the combination irrigation intervals every 3 days with compost treatment, respectively. The variation in branches number/plant between maximum and the minimum values reached to 100%.

Data shown in Table 6 indicated that, the combination between irrigation intervals every 3 days and NPK treatment gave the highest mean value of flowers heads number (25.00/plant),while the combination between irrigation intervals every 9 days and compost+compost tea

treatment gave the lowest mean value (13.00/plant). The variation in flowers heads number/plant between maximum and the minimum values reached to 92.31%.

Concerning the interaction treatments, it can be noticed that the combination between irrigation intervals every 3 days and compost+compost tea+mycorrhiza treatment resulted in the maximum mean value of seed yield (23.40 g/plant) while the interaction between irrigation intervals every 9 days and compost+compost tea treatment gave the lowest one (13.00 g/plant). The variation in seed yield (g/plant) between maximum and the minimum values reached to 78.49%.

The obtained results indicated the favorable effect of organic and bio fertilizers which reduce the harmful effect of water stress through their effect on improving the soil texture. The structural improvement can encourage the plant to have a good root development by improving the aeration in the soil. The favorable effects of these fertilizers may be due to the role of organic material for continues supply of nutrients, which improve some physical properties of soil and increase water retention (Abd-Elmoez et al. 1995; Fliessbach et al. 2000).

Table 6. Effect the interaction treatments between irrigation intervals and fertilization on growth and yield of Silybum marianum Seed yield (g/plant)

Flowers heads no/plant

Branches no/plant

Plant height (cm) Fertilizer treatments Irrigation

intervals19.50 25.00 7.00 183.00 NPK 3 days18.70 19.00 5.00 170.00 Compost22.60 18.00 6.00 175.00 Compost+mycorrhiza20.33 21.00 8.00 177.00 Compost+compost tea 23.40 20.00 7.00 190.00 Compost+compost tea+mycorrhiza 18.55 20.00 8.00 174.00 NPK 6 days15.40 17.00 7.00 165.00 Compost19.60 18.00 7.00 167.00 Compost+mycorrhiza15.50 17.00 6.00 166.00 Compost+compost tea 17.90 19.00 8.00 175.00 Compost+compost tea+mycorrhiza 16.40 19.00 10.00 166.00 NPK 9 days13.11 15.00 7.00 158.00 Compost14.50 14.00 9.00 163.00 Compost+mycorrhiza13.65 13.00 8.00 170.00 Compost+compost tea 16.80 18.00 9.00 174.00 Compost+compost tea+mycorrhiza 1.067 1.301 1.288 1.126 LSD at 5% Table 7. Effect irrigation intervals on silymarin content (mg/g seed) of Silybum marianum Irrigation Intervals Silychristin Silydianin Silybin A Silybin B Isosilybin A Isosilybin B Total 3 days 17.952 11.182 11.092 18.576 7.216 2.814 68.832 6 days 18.584 12.032 12.086 19.332 7.538 3.078 72.65 9 days 22.028 13.352 14.776 23.34 8.734 3.184 85.414 Table 8. Effect of fertilizer treatment on silymarin content (mg/g seed) of Silybum marianum Fertilizer treatments Silychristin Silydianin Silybin A Silybin B Isosilybin A Isosilybin B Total NPK 19.62 11.76 12.61 20.82 7.64 3.07 75.52 Compost 19.49 13.10 12.35 20.00 7.92 3.09 75.95 Compost+mycorrhiza 20.49 12.53 13.35 21.48 8.21 2.79 78.85 Compost+compost tea 19.37 12.34 12.80 20.27 8.18 3.36 76.32 Compost+compost tea+ mycorrhiza 18.34 11.22 11.96 19.51 7.20 8.47 76.7


Table 9. Effect the interaction treatments between irrigation intervals and fertilization on silymarin content (mg/g seed) of Silybum marianum

Total Isosilybin B

Isosilybin A

Silybin B

Silybin A Silydianin Silychristin Fertilizer treatments Irrigation

intervals 71.74 3.02 7.49 19.45 11.66 11.48 18.64 NPK 3 days 69.64 2.95 7.26 18.46 11.02 11.83 18.12 Compost 64.86 2.03 6.98 17.70 10.79 10.34 17.02 Compost+mycorrhiza 72.00 3.38 7.57 19.14 11.38 12.03 18.50 Compost+compost tea 65.92 2.69 6.78 18.13 10.61 10.23 17.48 Compost+compost tea+mycorrhiza 72.24 3.29 7.33 18.74 11.71 12.80 18.37 NPK 6 days 68.96 2.69 7.22 18.51 11.30 11.30 17.94 Compost 75.95 3.13 8.15 20.21 12.55 12.39 19.52 Compost+mycorrhiza 71.3 2.92 7.63 19.35 12.48 10.81 18.11 Compost+compost tea 74.8 3.36 7.36 19.85 12.39 12.86 18.98 Compost+compost tea+mycorrhiza 82.58 2.89 8.11 24.27 14.47 10.99 21.85 NPK 9 days 89.24 3.64 9.28 23.02 14.74 16.16 22.40 Compost 96.29 3.20 9.50 26.54 17.25 14.87 24.93 Compost+mycorrhiza 85.64 3.77 9.33 22.32 14.54 14.17 21.51 Compost+compost tea 73.32 2.42 7.45 20.55 12.88 10.57 19.45 Compost+compost tea+mycorrhiza

Silymarin content

Data tabulated in Tables 7, 8 and 9 indicated that total silymarin content (mg/g seed) ranged from 64.86 to 96.29 mg/g. The main constituent of silymarin were Silybin B (17.70-26.54 mg/g) followed by Silychristin (17.48-24.93 mg/g). In this connection, dried extracts of milk thistle seeds contain approximately 60% silymarin, where silymarin consists of four flavonolignans of silybinin (~ 50 to 60%), isosilybinin (~ 5%), silychristin (~ 20%) and silydianin (~ 10%) (Burgess, 2003). (Ibrahim et al. 2007) found that the concentration and total yield of six silymarin compounds showed wide variations between lines, varieties and generations ranged from 11.92 to 62.85 mg/g seed and between 329.8 to 2121.3 mg/plant, respectively. Six silymarin compounds: silychristin, silydinin, silybin A, silybin B, isosilybin A and isosilybin B were detected in the extract of all tested treatments. These results were in agreement with (Ibrahim et al. 2007).

Irrigation intervals Data tabulated in Table 7 show that, the mean values of

total Silymarin content (mg/g seed) were 68.83, 72.65 and 85.41 mg/g were obtained as a result of irrigation intervals at 3, 6 and 9 days, respectively.

Silybin B followed by silychristin were the main components of silymarin. The maximum mean values of Silybin B (23.34 mg/g) and Silychristin (22.03 mg/g) were observed as a result of irrigation intervals every 9 days.

Drought stress increases the secondary products percentage of more medicinal and aromatic plants, because in case of stress, more metabolites are produce in the plants and substances prevent from oxidization in the cells, but secondary products content reduce under drought stress, because the interaction between the amount of the secondary products percentage and mass production is consider important as two components of the secondary products content and by exerting stress, increases the secondary products percentage but mass production decreases by the drought stress, therefore secondary products content reduces. The data from (de Abreu and

Mazzafera 2005) showed that also the total amount of some secondary plant products per plant indeed is significantly higher in plants grown under drought stress than in those cultivated under normal conditions. Although stressed plants had been quite smaller, the product of biomass and substance concentration yields in a 10% higher amount of phenolic compounds; however, the total content of betulinic acid was nearly the same in plants when grown under drought stress or under standard conditions. Also the studies published by Nogues et al. (1998), who found a massive increase of phenolic compounds in stressed peas, allow calculating the overall yield of the related substances. Despite the fact that the total biomass of pea plants grown under drought stress is just about one third of those cultivated under standard condition, the overall amount of anthocyanins (product of biomass and anthocyanin concentration) is about 25% higher in the stressed plants. Apart from that, the overall yield of total flavanoids was nearly the same in Pisum sativum plants grown under drought stress or under non-stress conditions.

Fertilizer treatments Data tabulated in Table 8 indicated the effect of

different fertilizer treatments on silymarin content (mg/g). Total silymarin content ranged from 75.52 to 78.85 mg/g. Compost+mycorrhiza treatment gave the maximum mean values of total silymarin content (78.85 mg/g) followed by Compost+compost tea+mycorrhiza treatment which gave 76.70 mg/g. The highest mean values of Silybin B (21.48 mg/g) and Silychristin (20.49 mg/g) were obtained as a result of compost+mycorrhiza treatment compared with other treatments.

As for the favorable effect of applying organic and/or bio fertilizers on silymarin content may be due to effect of these fertilizers on accelerating metabolism reactions as well as stimulating enzymes. Application of bio fertilizers and compost significantly improved secondary products such as essential oil, rutin and coumarin (El-Sherbeny et al. 2007 a, b). Variations in plant growth and active principles in mycorrhizae inoculated plants have been reported for

5 (1): 22-29, May 2013

28

many other medicinal plants (Sailo and Bagyara 2005; Copetta et. al. 2006).

Interaction treatments It can be noticed that compost+ mycorrhiza treatment

under 9 days irrigation intervals gave the maximum value of total silymarin content (96.29 mg/g) followed by compost treatment under the same irrigation intervals which gave 89.24 mg/g (Table 9). The lowest value of Sylimarin content (64.86 mg/g) was obtained as a result of compost+mycorrhiza treatment under 3 days irrigation intervals.

Moreover, the highest values of Silybin B (26.54 mg/g) and Silychristin (24.93 mg/g) were observed as a result of compost+ mycorrhiza treatment under 9 days irrigation intervals. In this respect, mycorrhiza fungi play a critical role in interest cycling and ecosystem function. They improve plant growth and survival through a mutuality relationship in which photosynthates are exchanged for increased access to water and nutrients (Kernaghan 2004). These effects may be played an important role to increase the secondary metabolites accumulation.

CONCLUSION

All presented data indicated that all studied growth and yield characters were significantly affected by the duration of irrigation intervals also organic and bio fertilizer showed a primitive effect on growth and yield characters. The interaction between irrigation intervals and fertilizer treatments has a clear considerable effect on growth and yield characters. Organic and bio fertilizers can reduce the harmful effect of water stress.

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Study of altitude and selection on fiber biometry properties of Fagus orientalis Lipsky

ZOHREH ZOGHI1,♥, DAVOUD AZADFAR1, ALI KHAZAEIAN2

1Department of Forest Ecology, Faculty of Forest Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran. Tel./fax.: +98-171-2245882, ♥e-mail: [email protected]

2Department of Wood and Paper Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.

Manuscript received: 27 January 2013. Revision accepted: 30 April 2013.

Abstract. Zoghi Z, Azadfar D, Khazaeian A. 2013. Study of altitude and selection on fiber biometry properties of Fagus orientalis Lipsky. Nusantara Bioscience 5: 30-34. This research reports to the influence of altitude above sea level and selection on fiber biometry of beech wood (Fagus orientalis Lipsky). In this research, six trees in 550 MASL (three plus trees and three non-plus trees) and six trees in 850 MASL (three plus trees and three non-plus trees) were selected. One sample from each tree was gotten with increment borer at breast height. Fiber characteristics such as fiber length, fiber diameter, lumen width, and two wall thicknesses were measured in Franklin method. Slenderness ratio, flexibility ratio and rankle ratio were estimated. The results showed that the altitude had on significant effect on fiber length, lumen width, wall thickness and biometry coefficients. Tree quality has significant effect on fiber length, lumen width, and wall thicknesses and biometry coefficients. Fiber length, lumen width, slenderness ratio and flexibility ratio in plus trees were more than non-plus tree.

Key words: Fagus orientalis, fiber, biometry properties, plus trees.

Abstrak. Zoghi Z, Azadfar D, Khazaeian A. 2013. Studi ketinggian dan seleksi sifat-sifat biometri serat Fagus orientalis Lipsky. Nusantara Bioscience 5: 30-34. Penelitian ini melaporkan pengaruh ketinggian di atas permukaan laut dan seleksi biometri serat dari kayu beech (Fagus orientalis Lipsky). Dalam penelitian ini dipilih 6 pohon dari ketinggian 550 m dpl. (3 pohon unggul dan 3 pohon non-unggul) dan 6 pohon dari ketinggian 850 m dpl (3 pohon dan 3 pohon non-unggul). Satu sampel dari setiap pohon diperoleh dengan alat pengebor pada ketinggian dada. Sifat-sifat serat seperti panjang serat, diameter serat, lebar lumen, dan ketebalan dua dinding diukur dengan metode Franklin. Rasio kelangsingan, rasio kelenturan dan rasio rankle dihitung. Hasil penelitian menunjukkan bahwa ketinggian berpengaruh signifikan terhadap panjang serat, lebar lumen, ketebalan dinding dan koefisien biometri. Kualitas pohon berpengaruh signifikan terhadap panjang serat, lebar lumen, dan tebal dinding dan koefisien biometri. Panjang serat, lebar lumen, rasio kelangsingan dan rasio kelenturan pada pohon unggul lebih tinggi dari pada pohon non-unggul.

Kata kunci: Fagus orientalis, serat, sifat biometri, pohon unggul.

INTRODUCTION

The wood properties vary as a result of variation in fiber morphology within each annual ring formed, between trees and between stands (Zobel and van Buijtenen 1989). Wood quality characteristics can be influenced by both tree growth condition and genetic factors (Jyske 2008; Gaspar 2009). Wood anatomical structure relates to wood product properties like flexibility, plasticity, resistance, and optical features (Panshin and Zeeuw 1980; Zhang 1997; St-Germain and Krause 2008). Fiber length, lumen size and cell wall thickness have influence on the rigidity and strength properties (Oluwafemi and Sotannde 2007).

Plus tree selection is one of the first steps and used method of obtaining material for forest tree improvement programs (Zobel and Talbert 1984; Changtragoon 1996). Plus trees are phenotypes judged but not proved by test to be unusually superior in some quality and quantity, eg growth rate, disirable growth habit, high wood density and

exeptional apparent resistance to disease and insect attack (Nieuwenhuis 2000).

Regarding to wood economic importance and its usage on human life and limitation of natural recourses, determination of wood quality and appropriate application for suitable usage is necessary. This is dependent on identification of wood physical and anatomical properties (Doosthosseini and Parsapajouh 1996) and finding the relations between environmental and genetic factors on them. Some studies carried out to determine wood fiber properties of beech trees (Fagus orientalis Lipsky; Figure 1) and effect of them on strength properties (Akgul and Tozluoglu 2009). They found that utilization of juvenile woods on fiber production, can have contribution on raw material supply. In previous studies were found the latitude and altitude has major effects on variability in wood properties within species and, could have impact on juvenile wood rate production as well (Panshin and de Zeeuw 1980).

ZOGHI et al. – Fiber biometry properties of Fagus orientalis

31

Figure 1. Beech tree (Fagus orientalis Lipsky): A. Flowers, B. Fruit. (photos: from many sources).

Related studies can also provide knowledge and guidance for Kiaei (2011) reported that altitude and height of tree has effect on wood density and fiber biometry properties of hornbeam-Carpinus betulus (L.). With increase of altitude from sea level, the wood density, cell wall thicknesses and rankle ratio were increased and the fiber length, fiber diameter, fiber lumen diameter, slenderness ratio and flexibility ratio values were decreased. Varshoie et al. (2006) found that influence of altitude on fiber thicknesses of beech trees is significant however he did not find significant relation between environmental factor and other biometry properties. On the other study altitude did not affect on fiber length (Hosseini 2006). Ishiguri et al. (2007) suggested that the basic density of core wood is a very important factor for the selection of a plus tree in tree breeding for wood quality. Gaspar and et al. (2009) studied the consequences of selection on wood quality traits of Pinus Pinaster. They concluded that genetic selection based on growth will not result in a decrease of wood density, will not affect the occurrence of spiral grain, and is possible to obtain an increase in the radial modulus of elasticity. They suggest that selection for growth will probably not affect negatively the wood properties at future.

The wood of oriental beech is heavy, hard, strong and highly resistant to shock. It is one of the most important commercial woods in Iran. Oriental beech wood use as particleboard, furniture, flooring veneer, mining poles

(props), railway tiles and paper (Kandemir and Kaya 2009). In this study were investigated fiber and biometry properties of beech wood and effect of altitude and selection on them because we can identify application of wood with knowledge about wood physical and anatomical properties, and we can find a way for operating silviculture programs for more genetic conservation and improvement and extension of generation of suitable trees. This is provided production plus quality wood in natural forest.


Site study Iranian beech forests are located on the northern slopes

of Alborz Mountains, Hyrcanian forests, within an altitude of about 600-2000 m.a.s.l. They assemble a forest strip of 700 km length, located in three provinces of Guilan, Mazandaran and Golestan (Salehi Shanjani et al. 2011).

This study was conducted at Shast Kalate forest, at the Gorgan University of Agricultural Sciences and Natural Resources on the Golestan province. It is located in northern Iran (36° 41’ to 36° 45’ northern latitudes and 54° 20’ to 54° 24’ eastern longitudes) with an area of about 3716 ha and an altitude ranging from 100 to 1000 m above sea level (Figure 2).This mixed deciduous forest is covered different forest community such as Zelkova-Quercetum, Parrotio-Carpinetum, Fageto-Carpinetum and Fagetum

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ZOGHI et al. – Fiber biometry properties of Fagus orientalis

33

Table 1. The result of the analysis of variance for fiber properties of beech trees Feature source F sig

Fiber length

Corrected Model 4.84 0.00** Altitude 1.45 0.23ns selection 11.54 0.00** Altitude*Selection 1.53 0.22 ns

Fiber diameter

Corrected Model 2.81 0.04* Altitude 0.03 0.87 ns selection 1.23 0.27 ns Altitude*Selection 7.17 0.01**

Fiber lumen width

Corrected Model 4.53 0.00** Altitude 0.02 0.89 ns selection 13.56 0.00** Altitude*Selection 0.00 0.97 ns

2 wall thicknesses

Corrected Model 9.47 0.00** Altitude 0.00 0.95 ns selection 19.88 0.00** Altitude*Selection 8.52 0.00**

Slenderness ratio


Flexibility ratio


Rankle ratio


Note: *: Significant differences (level of significance p < 0.05); **: Significant differences (level of significance p < 0.01); ns: Not significant differences between the treatments; P > 0.05 Table 3. The mean of value (±standard deviation) belong to morphological properties of beech fibers

Rankle ratio

Flexibility ratio

Slenderness ratio

Properties

3.15±1.63 27.32±9.03 78.39±15.15 550 Altitude (m) 3.15±1.67 27.36±9.43 79.58±17.77 850 2.86±1.45 28.92±9.17 81.01±17.29 Plus trees Phenotype

quality 3.44±1.78 25.76±9.03 76.96±15.46 Non-plus trees

Discussion The fiber morphology affects the processing and

properties of both lumber and paper (Seth 1990; Kibblewhite and Bawden 1991; Skinnarland et al. 1995;

Seth et al. 1997; Vahey et al. 2007). Some factors such as soil, climate, and altitude and forest management lead to appear differences on wood properties of timber of same species (Doosthosseini and Parsapajouh 1997). The hardwood plant species had significant difference on wood density, fiber properties and mechanical strength (Kiaei and Samariha 2011).

Even though a difference in altitude of about 300 m could not seriously have effect fiber morphology and biometry coefficients in beech trees, it seems that altitude from sea level should play a positive role with beech trees when the difference is greater than it (Hosseini 2006). St-Germain and Krause (2008) found that latitude (along a 500 km transect) had no effect on tracheid length. Hosseini (2006) observed altitude in the range of about 500 m no important effect on beech fiber length.

In this investigation plus trees had long fiber, small fiber diameter, wide fiber lumen and thin wall thickness. The value of slenderness ratio and flexibility ratio in plus trees was bigger than non-plus trees but the value of rankle ratio in plus trees was lower than non-plus trees. The beech plus trees are superior on phenotype in comparison with beech non-plus trees, they have good stem form like stem straightness, non-twisting bole, non-undulating, more clear bole height (CBH), diameter polarity and crown polarity it cause to increase fiber length and decrease fiber diameter. The species with higher lengths, small diameter, thin wall cell and large cell lumen are more desirable for paper formation and strength (Monteoliva et al. 2005; Gaspar 2009). Regard to beech plus trees have been selected to reach suitable industrial wood but results are shown that non-plus trees in comparison with plus trees have more desirable strength properties therefore they are can be used on fiber board production and wood plus trees suitable for fiber plate, rigid cardboard production.

Regard to stem form is one of easiest and quickest ways to improve wood quality, because it can be controlled both genetically and silvicultural and because gains can be substantial and rapid (Zobel and Talbert 1984). Selection of plus trees do for changing some characteristics like growth rate, stem form, resistance to disease, branching habit and wood structure. It is also provided for reproduction of desirable characteristics. When this characteristics are controlled genetically, they can be affected on mean of gain of selected trees (Mahoney and Fins 2001) but some trees that have a high growth rate or good stem form do not always produce industrially desirable wood (Ishiguri et al. 2007). The differences in wood properties among provenances, families and/or individual trees provide an opportunity for breeding programmers to select superior trees for solid wood production (Gapare et al. 2012).

Table 2. The mean of fiber dimension (±standard deviation) in 2 different altitudes and 2 qualities

2*Wall thicknesses (µm) Lumen width (µm) Fiber diameter (µm) Fiber length (µm) Properties

14.96±2.42 5.68±2.15 20.64±2.60 1590.52±244.16 550 Altitude 14.97±2.58 5.70±2.24 20.67±2.73 1611.07±217.28 850 14.56±2.47 5.99±2.21 20.54±2.69 1629.83±214.14 Plus trees Quality 15.37±2.47 5.39±2.13 20.76±2.63 1571.76±243.91 Non-plus trees

5 (1): 30-34, May 2013

34

Since fiber morphology are usually highly inherited (Boyle et al. 1987; Longman 1993; Hylen 1999; Zubizarreta-Gerendiain et al. 2008) and the breeding programs for wood quality has a strong potential (Ishiguri et al. 2007), select of plus trees with desirable wood traits should be considered in tree improvement programs.

CONCLUSION

This study demonstrates that altitude of about 300 m could not seriously have effect on fiber morphology and biometry coefficients in beech trees. Selection of plus trees without examination of wood properties may be useless for improving programs. The results from this study suggest that identification of beech plus trees have to do with considering phenotype and desirable wood properties depend on final use. It is necessary to do progeny test to prove heritage of wood properties to gene conservation stands as well. also, after they use as reproductive material for proper use.

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Coral disease distribution at Ras Mohammed and the Gulf of Aqaba, Red Sea, Egypt

MOHAMMED SHOKRY AHMED AMMAR1,♥, FEKRY ASHOUR2, HODA ABDELAZIM2 1Department of Hydrobiology, National Institute of Oceanography and Fisheries, P.O. Box 182, Suez, Egypt. Tel. +20 111 1072982,

Fax. +20 623360016, E-mail: [email protected] 2National Institute of Oceanography and Fisheries, Suez, P.O. Box 182, Egypt

Manuscript received: 27 January 2013. Revision accepted: 5 May 2013.

Abstract. Ammar MSA, Ashour F, Abdelazim H. 2013. Coral disease distribution at Ras Mohammed and the Gulf of Aqaba, Red Sea, Egypt. Nusantara Bioscience 5: 35-43. Six sites along the Gulf of Aqaba and Ras Mohammed, Red Sea, Egypt were studied for coral disease distribution relative to environmental stress. These sites are (i) South Taba, (ii) South Nuweiba, (iii) Canyon, (iv) Eel Garden (at Dahab), (v) Shark Observatory and (vi) Yolanda. Number of coral diseases ranges from 6 diseases at site 4 (Eeel Garden) to 12 diseases at site 3 (Canyon). The site having the lowest number of coral diseases (site 4) is characterized by the highest percentage cover of coral diseases (24%). The coral disease atramentous necrosis attained the highest percentage cover in all sites (5, 5, 6, 6, 2 and 3%) in sites 1-6 respectively. A total of 16 diseases were reported being distributed in the following order in sites 1-6: 9, 9, 12, 6, 8 and 7 respectively. The coral disease atramentous necrosis is the most widely distributed one being found in all 6 sites followed by dark spots disease and ulcerative white spots being reported in 5 sites. The disease that is least distributed is the white tips being reported in site 5 only. The most commonly distributed disease (atramentous necrosis) infected six corals in site 1, two corals in site 2, nine corals in site 3, two corals in site 4, five corals in site 5 and five corals in site 6. However, the least commonly distributed disease (white tips) infected only two corals (Acropora humilis and Millepora dichotoma). Site 1, having Cyphastrea serialia being infected with highest number of diseases is characterized by the maximum metal concentrations of Zn, Cd, Pb and Ni in water and highest metal concentrations for Cu, Zn and Pb in sediments. Site 2, having M. dichotoma being infected with the highest number of diseases, is characterized by the highest Cu concentration in water. Site 4, having fewer number of coral diseases and highest percentage disease cover attained the highest levels of Cd and Ni in sediments.

Key words: Coral disease, distribution, Ras Mohammed, Gulf of Aqaba, Red Sea

Abstrak. Ammar MSA, Ashour F, Abdelazim H. 2013. Distribusi penyakit karang di Ras Mohammed dan Teluk Aqaba, Laut Merah, Mesir. Nusantara Bioscience 5: 35-43. Enam situs di sepanjang Teluk Aqaba dan Ras Mohammed, Laut Merah, Mesir dipelajari untuk mengetahui distribusi relatif penyakit karang terhadap tekanan lingkungan. Lokasi yang diteliti adalah (i) South Taba, (ii) South Nuweiba, (iii) Canyon, (iv) Eel Garden (di Dahab), (v) Shark Observatory dan (vi) Yolanda. Jumlah penyakit karang berkisar dari 6 penyakit di lokasi 4 (Eeel Garden) hingga 12 penyakit di lokasi 3 (Canyon). Lokasi yang memiliki jumlah penyakit karang terendah (lokasi 4) ditandai dengan persentase penutupan penyakit karang tertinggi (24%). Penyakit karang nekrosis atramentous mencapai persentase penutupan tertinggi di semua lokasi (5, 5, 6, 6, 2 dan 3%) secara berturut-turut dari lokasi 1-6. Sebanyak 16 penyakit dilaporkan terdistribusi dengan urutan dari lokasi 1-6 secara berturut-turut sebagai berikut: 9, 9, 12, 6, 8 dan 7. Penyakit karang nekrosis atramentous merupakan penyakit yang paling luas distribusinya yang ditemukan di semua ke-6 lokasi, diikuti oleh penyakit bintik-bintik gelap dan bintik-bintik putih ulseratif yang dilaporkan pada 5 lokasi. Penyakit yang paling sempit distribusinya adalah pucuk putih yang dilaporkan dalam 5 lokasi. Penyakit yang paling luas distribusinya (nekrosis atramentous) menginfeksi enam terumbu karang di lokasi 1, dua karang di lokasi 2, sembilan karang di lokasi 3, dua karang di lokasi 4, lima karang di lokasi 5 dan lima karang di lokasi 6. Namun, penyakit yang paling sempit distribusinya (pucuk putih) hanya menginfeksi dua karang (Acropora humilis dan Millepora dichotoma). Lokasi 1, terdapat Cyphastrea serialia yang terinfeksi penyakit dengan jumlah paling tinggi ditandai dengan konsentrasi maksimum logam Zn, Cd, Pb dan Ni dalam air dan konsentrasi logam tertinggi untuk Cu, Zn dan Pb dalam sedimen. Lokasi 2, terdapat M. dichotoma yang terinfeksi penyakit dengan jumlah tertinggi, ditandai dengan konsentrasi Cu tertinggi dalam air. Lokasi 4, memiliki lebih sedikit jumlah penyakit karang dan persentase penutup penyakit tertinggi mencapai tingkat tertinggi Cd dan Ni dalam sedimen.

Kata kunci: Penyakit karang, distribusi, Ras Mohammed, Teluk Aqaba, Laut Merah

INTRODUCTION

Coral disease is defined as an abnormal condition of an organism that impairs organism functions, associated with specific symptoms and signs (ICRI/UNEP-WCMC 2010). It may be caused by external factors, such as infectious disease, or it may be caused by internal dysfunctions. Coral

disease outbreaks are having a significant, negative impact on the structure and appearance of coral reefs, and have contributed to unprecedented declines in live coral cover and productivity of coral reef ecosystems upon which many millions of people depend (Galloway et al. 2009). The same authors concluded that several diseases are playing an increasingly important role in controlling coral population

5 (1): 35-43, May 2013

36

size, diversity and demographic characteristics.. Large scale disease outbreaks have already fundamentally altered the structure of reef communities in the Caribbean (Harvell et al. 2004). Research on the causes of coral disease has increased in recent years, especially in terms of identifying the pathogens involved (Harvell et al. 2007). Most biotic coral diseases are believed to be related to infection by one or a group of pathogens (Sokolow 2009). A host of contributing microorganisms (Richardson and Aronson 2000) and macroparasites such as ciliates (Cróquer et al. 2006) have been identified as possible causal agents; however, little is currently known about the involvement of viruses (Sokolow 2009). Research on the causes of coral disease has increased in recent years, especially in terms of identifying the pathogens involved (Harvell et al. 2007). Knowledge of organisms that transmit pathogens from a reservoir to a host (vectors), the mechanisms by which coral disease is transmitted between organisms (vector pathways), and natural reservoirs of coral disease is far from complete (ICRI/UNEP-WCMC 2010). Growing evidence suggests that environmental and anthropogenic stressors are linked with coral disease and mortality in complex ways (Harvell et al. 2007). Examples of of those stressors are nutrient enrichment (Garren et al. 2008), ocean acidification (Sokolow 2009), algal competition (Aronson and Precht 2006), irradiance (Boyett et al. 2007) and loss of biodiversity (Keesing et al. 2010).

Coral disease identification is often based on visual cues observed in the field or from photographs. Such techniques have been shown to be insufficient for making coral disease because different causes of disease can result in similar obvious manifestations of disease, or progress from showing the signs of one disease to showing those of another (Ainsworth et al. 2007). Ammar (2012) provided a guide to coral diseases in the northern Red Sea, Egypt. Laboratory analyses of samples to identify the microbio-logical factors accompanying the disease manifestations, such as the presence or absence of certain pathogens, are therefore necessary to support accurate disease diagnosis and accurate disease identifications (Ainsworth et al. 2007).

The purpose of the study is to quantify the coral diseases in many areas of the Gulf of Aqaba and Ras Mohammed (South Sinai), Egypt. In addition, the environmental drivers of disease, as well as understanding the coral’s ability to resist the disease are studied. A data based on coral diseases in the area will be established, this will help using coral diseases as indicators of environmental impacts and acting to remove or minimize these impacts. Removing or minimizing these impacts will improve the coral reef environment, in turn help to increasing fish stocks, tourist attraction, improving the national income, the economic, scientific and medical values and conserving the marine biodiversity.


Six sites along the Gulf of Aqaba and Ras Mohammed, Red Sea, Egypt (Figure 1, Table 1) were studied for coral diseases.

Table 1. Latitudes and longitudes of the study sites Sites Latitudes Longitudes 1. South Fanar village (South

Taba) 29°20.170 ̀N 034°45.767 ̀E

2. South Nuweiba (2 km south of Nuweiba harbor)

28° 57.521 ̀ N, 034° 38.516 ̀ E

3. Canyon (north Dahab) 28° 33.277 ̀ N, 034° 31.235 ̀ E 4. Eel Garden (at Dahab) 28° 30.297 ̀ N, 034° 31.171 ̀ E 5. Shark Observatory (at Ras

Mohammed) 27° 43.921 ̀ N, 034° 15.560 ̀ E

6. Yolanda (at Ras Mohammed)

27° 43.715 ̀ N, 034° 15.383 ̀ E

Coral diseases were quantified as percentage cover

relative to the bottom cover. SCUBA diving and the camera frame (as a quadrat) were used for surveying the coral diseases. Ten frames, one meter intervals and one meter from the object were surveyed along a transect fixed horizontally along the reef contour at the depths reef flat, 1 m, 5 m, 10 m, 15 m, 20 m or till the end limit of coral growth at each of the studied sites. A FinePix F50, 12 Mega Pixels Digital Camera, was used for taking a series of underwater photos to help identification of species and coral diseases. The computer software Photogrid 1.0 beta Acad was used for ecological analysis of digital photographs for coral diseases.

Coral disease pathogen identification was achieved using ICRI/UNEP-WCMC (2010), Raymundo et al. (2008), Rosenberg et al. (2007).

Disease definition and disease types Only clear and unequivocal signs of disease were

recorded. Coral disease was also carefully distinguished from coral bleaching (Brown 1997), which superficially can look like disease. To make a disease determination, observers looked for active tissue necrosis. Often this was accompanied by bared skeleton, mucus production and partial disintegration of polyps. Blemishes, slight discolorations and small, cryptic examples of disease were not scored. We chose characteristics that were as pathognomicas possible for underwater determinations. Anchor scrapes, parrot fish bites, predatory snail wounds, etc were not scored as diseases but as causative agents.

Quality assurance/quality control After the first survey of sites, the underwater survey

lines were taken up, and then reset and surveyed once again. In addition, a video tape of lines were done.


Number and percentage cover of coral diseases, healthy corals and associated biota in each of the studied sites are shown in Table 2, while the infected coral species by different diseases are found in Tables 3. Number of coral diseases ranges from 6 diseases at site 4 (Eeel Garden) to 12 diseases at site 3 (Canyon). However the site having the lowest number of coral diseases (site 4-6 diseases)

AMMAR et al. – Coral disease distribution at Red Sea, Egypt

37

Figure 1. Map of the studied sites. 1. South Fanar village (South Taba), 2. South Nuweiba (2 km south of Nuweiba harbor), 3. Canyon (north Dahab), 4. Eel Garden (at Dahab), 5. Shark Observatory (at Ras Mohammed), 6. Yolanda (at Ras Mohammed)

is characterized by the highest percentage cover of coral diseases (24%) Those diseases are sediment damage, dark spots, coral neoplasia, ulcerative white spots, coral hyperplasia and atramentous necrosis. Sites 5 (Shark Observatory) and 6 (Yolanda) are characterized by the highest amount of percentage healthy corals (85% and 83% respectively) while the lowest value is found in site 4 (16%). The highest percentage cover of algae/sea grasses (22%) is found in site 4 while the lowest percentage cover of each of algae/seagrasses, macroborers, and sediments is found in site 5.

The coral disease atramentous necrosis attained the highest percentage cover in all sites (5, 5, 6, 6, 2 and 3%) in sites 1-6 respectively. Diseases having lowest percentage cover are black band disease (site 1), white band disease (site 2), pigmentation response (site 3), coral hyperplasia (sites 4, 5) and black band (site 6). A total of 16 diseases were reported being distributed in the following order in sites 1-6: 9, 9, 12, 6, 8 and 7 respectively. The coral disease atramentous necrosis is the most widely distributed one being found in all 6 sites followed by dark spots and ulcerative white spots being reported in 5 sites. The disease

that is least distributed is the white tips being reported in site 5 only. However, each of the black band, white spots, white band, pigmentation response, sediment damage and rapid wasting is reported in 2 sites only. The most commonly distributed disease (atramentous necrosis) infected six corals in site 1, two corals in site 2, nine corals in site 3, two corals in site 4, five corals in site 5 and five corals in site 6. However, the least commonly distributed disease (white tips) infected only two corals (Acropora humilis and Millepora dichotoma).

It is observed that the coral disease ulcerative white spots is always associated with vermetidae predation in both earlier and later stages of the disease, and in many cases with Tridacna boring in later stages of the disease. However, vermetidae predation is also associated with tissue discolouration (non white pigmentation response) while Drupella predation is associated with skeletal eroding band. The coral disease tissue coral neoplasia is found only in site 4 having 4 percentage cover and infecting the two coral species Leptoseris incrustans and Favia speciosa.

3

4

1

2

6 5

PALESTINE

5 (1): 35-43, May 2013

38

Table 2. Percentage of coral diseases and other habitats in the studied sites

Sites

No.

of c

oral

di

seas

es

% c

oral

di

seas

es

% h

ealth

y co

rals

% d

ead

cora

ls

% a

lgae

/ se

a gr

asse

s

% m

acro

-bo

rers

% se

di-

men

ts

% o

ther

s

1. South Fanar village (South Taba) 9 15 30 20 10 8 12 5 2. South Nuweiba (2 km south of Nuweiba harbor) 9 20.5 23 19.5 13 9 13 2 3. Canyon (north Dahab) 12 22 35 16 7 6 9 3 4. Eel Garden (at Dahab) 6 24 16 15 22 2 20 1 5. Shark Observatory (at Ras Mohammed) 8 7.75 85 2.5 0.5 1.25 0 3 6. Yolanda (at Ras Mohammed) 7 8 83 3 1 2 1 2 Table 3. Coral diseases at at Ras Mohammed and the Gulf of Aqaba, Red Sea, Egypt (site 1-6).

Sites Coral disease Infected coral species % disease cover Remarks

1. South Fanar village

1. Atramentous necrosis Goniastrea retiformisGoniastrea pectinata Cyphastrea serialia Platygyra lamellina Porites solida Millepora platyphylla

5 Mechanical breaking

2. Black band disease Goniastrea retiformis 0.4 3. Brown band disease Acropora nasuta

Favites flexusa0.5

4. Ulcerative white spots Porites solidaFavia speciosa Goniastrea retiformis

2.6 Vermetidaepredation Tridacna boring

5. White spots disease Echinopora gemmacea 0.5 6. Dark spots disease Cyphastrea serialia

Hydnophora exesa Porites solida Favites flexusa

2.5

7. White patches Favites flexusa 1 8. Skeleton eroding band Cyphastrea serialia

Siderastrea savignyana Platygyra lamellina

2 Drupella predation Drupella predation Drupella predation

9. White plague Cyphastrea serialiaGoniastrea retiformis

0.5

Total % disease cover

15

2. South Nuweiba

1. White plague Millepora dichotomaMillepora platyphylla

3.5 Overgrowth by Padina, Red filamentous algae, coralline algae

2. Atramentous necrosis

Cyphastrea serialiaMillepora dichotoma

5 Tridacna boring Mechanical breaking

3. Brown band disease Millepora dichotoma 2 4. White patches Cyphastrea serialia 2 Surface cyanobacteria 5. Dark spots disease Pavona cactus

Montipora verrucosa2

Gastropod boring 6. White band disease Stylophora pistillata 1 7. Ulcerative white spots Porites soloda 1.5 8. Coral hyperplasia Millepora dichotoma 1.5 Mmechanical breaking 9. Partial bleaching Favia favus

Goniastrea retiformis2


20.5

3. Canyon 1. White plague Porites solida

2 Aggressive coralline algal overgrowthAggressive filamentous algal overgrowth Aggressive sponge overgrowth

2. Atramentous necrosis Goniasatrea pectinataGoniastrea retiformis Platygyra daedalia Lobophyllia corymbosa Favites flexusa Acropora tenuis Millepora platyphyla Millepora dichotoma Porites lutea

6 Mechanical breaking Tridacna boring Mechanical breaking Mechanical breaking Mechanical breaking Mechanical breaking Mechanical breaking Mechanical breaking Tridacna boring

3. Brown band Montipora informis 2 Gastropod drilling


39

Montipora verrucosaPorites lutea

Vermetidae predation

4. White patches Acropora validaAcropora hemprichi Acropora tenuis

3Drupella predation

5. Pigmentation response Stylophora pistillata 0.4 Ciliate infection 6. White spots disease Porites rus

Astreopora myriophthalma Porites solida

1.6Gastropod boring

7. Coral hyperplasia Montipora verrucosa 0.5 8. White band disease Porites lutea

Montipora verrucosa0.5

9. Partial bleaching Goniastrea retiformis 1 10. Sediment damage Favites flexusa 0.5 11. Skeletal eroding band Platygyra daedalea

Montipora informis Porites lutea

1.5Drupella predation

12. Rapid wasting Montipora tuberculosaPorites lutea Porites solida

3 Parrot fish predation Parrot fish predation Aggressive coralline algal overgrowth


22

4. Eel Garden 1. Sediment damage Leptoseris incrustansFavia speciosa

4 Aggressive red filamentous algal overgrowth

2. Dark spots disease Leptoseris incrustans 3 3. Coral neoplasia Leptoseris incrustans

Favia speciosa4 High sediment load

4. Atramentous necrosis Favia favusGoniastrea retiformis

6 Aggressive red filamentous algal overgrowth

5. Coral hyperplasia Psammocora haimeana 2 High sediment load 6. Ulcerative white spots Goniastrea retiformis 5 Total % disease cover

24

5. Shark Observatory

1. Atramentous necrosis Stylophora pistillataGoniastrea retiformis Porites lutea Acropora humilis Favia stelligera

2 Mechanical breaking Vermetidae predation Mechanical breaking

2. Pigmentation response Stylophora pistillataPorites solida

1 Gastropod boring Vermetid boring

3. Dark spots disease Porites solidaStylophora pistillata

0.5

4. Ulcerative white spots Goniastrea retiformisFavites flexusa

1

5. Coral hyperplasia Stylophora pistillata 0.25 Mechanical breaking 6. Partial bleaching Favites flexusa

Favia stelligera1

7. White tips Acropora humilisMillepora dichotoma

1 Vermetid boring, mechanical breaking

8. Rapid wasting Pavona explanulataEchinopora gemmacea

1 Aggressive coralline algal overgrowth


7.75

6. Yolanda 1. Dark spots disease Stylophora pistillataStylophora mamillata Goniastrea retiformis

1

2. Atramentous necrosis Stylophora mamillataStylophora pistillata Goniastrea retiformis Porites rus Pocillopora damicornis

3 Mechanical breaking Vermetidae predation Vermetidae predation Vermetidae predation Mechanical breaking

3. Ulcerative white spots Goniastrea retiformisPorites solida

1 Vermetedae predation

4. Black band disease Porites solida 0.25 5. Skeletal eroding band Porites lutea 0.5 Drupella predation 6. White plague Porites lutea

Porites solida Stylophorapistillata

1 Vermetedi Vermetedi Aggressive coralline algal overgrowth

7. White patches Favites abditaPorites rus Porites solida Stylophora mamillata

1.75 Parrot fish predation


8

5 (1): 35-43, May 2013

40

Figure 2. Several coral diseases at Ras Mohammed and the Gulf of Aqaba, Red Sea, Egypt. A. Ulcerative white spots, B. Dark spots disease, C. Coral tissue tumor, D. White band disease, E. Pigmentation response, F. Black band disease

Trace metals in water and sediments Total metal concentrations in seawater varied between

0.1 ppb for Cu and 2.51 ppb for Zn. Site 1, in the north, recorded the maximum metal concentrations for Zn, Cd, Pb and Ni . This may be due to the high pollution load from large cities and harbors like Aqaba and Elat. Site 2, south to Nuweiba, recorded highest Cu concentration, this may be due to pollution coming from Nuweiba harbour city. While, metal concentrations in sediments varied between 1.5 ppm for Cd and 20.79 ppm for Ni. Site 1 recorded the maximum metal concentrations for Cu, Zn and Pb. Site 4, in Dahab recorded the highest levels of Cd and Ni (Table 4). Table 4. Trace elements in surface water and sediments (ppb) Element Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Water Cu 0.26 0.28 + 0.22 0.13 0.11 0.09Zn 2.51+ 1.98 1.03 0.96 0.72 0.68Cd 1.56 + 1.11 0.73 0.72 0.66 0.61Pb 1.11 + 0.87 0.71 0,77 0.61 0.57Ni 0.87 + 0.66 0.57 0.49 0.39 0.41Sediment Cu 4.67 + 3.97 3.72 3.63 2.92 *Zn 11.33 + 7.27 6.25 6.71 5.28 *Cd 3.57 3.27 3.43 3.89 + 1.49 *Pb 17.74 + 17.55 16.76 16.91 12.46 *Ni 17.23 16.49 19.25 20.79 + 13.58 *Note: *Rocky bottom with no sediments

Discussion As it is obvious from the present study, the site having

the lowest number of coral diseases (site 4) is characterized by the highest percentage cover of coral diseases indicating space monopolization and outbreak of those diseases. The same site is characterized by the highest percent cover of sediments, suggesting that sedimentation may increase the percent coral diseases at the expense of disease number. This could be due to decreased coral mortality with increased sedimentation, decreasing the available substrate or space for diverse diseases. Decreased resistance of the host coral caused by adverse environmental conditions may increase opportunistic diseases (Harvell et al. 1999) leading to increased coral mortality (Haapkyla et al. 2009). Changes in the population size (e.g. percentage cover), growth and reproduction of a community’s primary producers (e.g. algae) and major framework builders will have impacts on the community. These changes are especially relevant given the longevous age structure of corals and, as compared to macroalgae, their relatively slow coral recruitment (Tougas and Porter 2002). This agrees with the results of the present study in which the highest percentage cover of algae/sea grasses is associated with the highest percentage cover of coral diseases (site 4), but the lowest percentage cover of each of algae/seagrasses, macroborers, and sediments is associated with the lowest percentage cover of coral diseases (site 5).

D E F

A B C


41

Jones et al. (2004) indicated that, Fluorescence in situ hybridisation (FISH) techniques and cloning, and analysis of the 16S rRNA genes from diseased coral tissue infected with atramentous necrosis, identified a mixed microbial assemblage in the diseased tissues particularly within the Alphaproteobacteria, Firmicutes and Bacteroidetes. In the present study, the presence of the coral disease atramentous necrosis attaining the highest percentage cover and infecting the highest number of corals in all sites, is associated with vermetidae predation in site 1, Tridacna boring, vermetidae in site 2, mechanical breaking, vermetidae in site 3, filamentous algal overgrowth in site 4, mechanical breaking, vermetidae in sites 5 and 6. This is an evidence that vermitidae predation, Tridacna boring and mechanical breaking may evoke growth of Alphaproteo-bacteria, Firmicutes and Bacteroidetes, promoting the growth of filamentous algae. Outbreak of the coral disease atramentous necrosis may be greately attributed to the terrestrial runoff caused by higher rainfall and in turn, decreased salinity (Harvell et al. 1999). This may have lead to increased stress on corals that may have reduced their immune responses, and/or increased virulence of pathogen (s) causing the disease. Decreased resistance of the host coral caused by adverse environmental conditions may also increase opportunistic diseases (Harvell et al. 1999). Results of the present study showed that the coral disease atramentous necrosis attained the highest percentage cover in all sites. This result, together with the fact that the present sites lie all in wadis being liable to terrestrial runoff , make the present results in aggrement with that of Harvell et al. (1999).

Lesions with signs that are similar to ulcerative white spots (UWS) can be caused by fish bites. Parrotfish lesions can be distinguished by the presence of skeletal damage, while the tubelip wrasse, Labrichthys unilineatus will remove tissue without damaging the skeleton. Arboleda and Reichardt (2010) stated that, the causative agent of the Indo-Pacific coral disease, Porites ulcerative white spot syndrome (PUWS), that affects Porites spp. and a few other coral genera has so far remained unidentified. In the present study, the association of the coral disease ulcerative white spots with vermetidae predation in both earlier and later stages of the disease, and in many cases with Tridacna boring in later stages of the disease is an evidence that vermetidae is a causative agents of the disease but Tridacna spp. could take the disease as a suitable substrate. In the present study, this disease infected Porites solida, Goniastraea retiformis, Favites flexusa and Favia speciosa.

Wooldridge (2010) indicated that coral's failure to prevent the division of zooxanthellae leads to ever-greater amounts of the photosynthesis-derived carbon to be diverted into the algae rather than the coral. This makes the energy balance required for the coral to continue sustaining its algae more fragile, and hence the coral loses the ability to maintain its parasitic control on its zooxanthellae leading to bleaching. In the present study, the white tips, which is somekind of bleaching, and infecting only the two species Acropora humilis and Millepora dichotoma, indicates that those two species are the most sensitive species that loses the ability to maintain its parasitic control on its

zooxanthellae. Lesions of tissue discolouration (non white pigmentation response) may be caused by borers, competitors, algal abrasion, fish bites, breakages, etc (Beeden et al. 2008). In the present study, the non white pigmentation response was associated with vermitidae predation) while skeletal eroding band was associated with Drupella predation.

Yamashiro et al. (2000) found coral neoplasia to be associated with the global coral bleaching event (1998). In the present study, the coral disease tissue coral neoplasia, being found only in site 4, was associated with high sediment load, low salinity due to fresh water coming from the adjacent tourist showers which are just close to the shore.

Sites 5 and 6, having the most healthy, rich, and nice reef slopes in the Red Sea, have their diseases restricted only on the reef flat. Those diseases of the reef flat are associated with mechanical breaking (due to trampling on the reef flat), vermetidae predation, gastropod boring, aggressive coralline algal overgrowth, Drupella predation and Parrot fish predation.

Diseases having lowest percentage cover are black band disease (site 1), white band disease (site 2), pigmentation response (site 3), coral hyperplasia (sites 4, 5), black band disease (site 6). However, Richardson (1998) indicated that the incidence and prevalence of black band disease may also increase when corals are stressed by sedimentation, nutrients, toxic chemicals and warmer-than-normal temperatures. Histopathological examinations of diseased tissue of white band disease (WBD) revealed basophilic ovoid bodies up to 40 μm (Peters et al. 1983). Electron microscopy of thin sections of the ovoid bodies revealed that they were composed of Gram-negative bacteria , suggesting that these bacteria may be the causative agent of the disease. Pigmentation response is considered a response of the coral host to a variety of stressors (e.g. unidentified pathogens, competition, predation, boring fauna, abrasion, etc.), suggesting that organism health is compromised (Raymundo et al. 2008).

White plague was reported in sites 3, 6. Richardson (1998) succeeded in isolating from diseased corals with white plague, a new species of Sphingomonas that infected healthy corals in laboratory experiments. Although the mechanism trigerring coral neoplasia or tissue coral neoplasia is still unknown and thought to be a genetic mutation that may be the result of environmental conditions (Yamashiro et al. 2000), tissue coral neoplasia disease in the present study ,being reported only in site 4, was associated with high sediment load, low salinity due to fresh water coming from the adjacent tourist showers which are just close to the shore. The disease in the present study was recognized as slightly hemispherical protuberances with fewer numbers of polyps per surface area, fewer zooxanthellae per polyp, finer skeletal structures than normal and reduced fecundity in coral neoplasia areas.

Infected corals relative to water and sediment quality As human populations continue to increase, nutrients,

terrigenous silt, pollutants and even pathogens themselves can be released into nearshore benthic communities (Raymundo et al. 2008). It was further discussed in that

5 (1): 35-43, May 2013

42

book that while the link between anthropogenic stress and disease susceptibility is currently poorly understood, one hypothesis is that coral disease is facilitated by a decrease in water quality, particularly due to eutrophication and sedimentation. Growing evidence suggests that environ-mental and anthropogenic stressors are linked with coral disease and mortality in complex ways (Harvell et al. 2007). Like, many benthic filter feeders, corals assimilate differentially certain amounts of solid metals, mainly through ingestion of contaminated particles (Madkour 2011). Ammar et al. (2005) concluded that the toxic effect of a certain metal on a coral may have the growth rate, in turn skeletal density, decreased with increasing metal concentration. This may foster the infection of the coral with a certain disease as well. Site 1 has 9 diseases infecting 12 coral species, of which Cyphastrea serialia is infected with highest number of diseases (atramentous necrosis, dark spots disease, skeletal eroding band and white plague). Site1 is characterized by the maximum metal concentrations of Zn, Cd, Pb and Ni in water and highest metal concentrations for Cu, Zn and Pb in sediments due to the high pollution load from large cities and harbors like Aqaba and Eilat. Site 2 has 9 diseases infecting 10 corals, of which Millepora dichotoma is infected with the highest number of diseases (white plague, atramentous necrosis, brown band disease and coral coral hyperplasia). Site 2 is characterized by the highest Cu concentration in water due to pollution coming from Nuweiba harbour. Site 3 has 12 diseases infecting 19 species (highest number of all sites), of which Porites lutea is infected with the highest number of diseases (atramentous necrosis, brown band disease, white band disease and skeletal eroding band). Site 4 has 6 diseases infecting 11 species, each of which is infected with only one disease except Favia speciosa and Goniastrea retiformis which are infected with 2 diseases for each one. Those fewer number of coral diseases in site 4 attained the highest percentage disease cover indicating space monopolization of those six diseases. This site attaind the the highest levels of Cd and Ni in sediments and highest percentage sediments. Site 5 has 8 diseases infecting 10 species, of which Stylophora pistillata is infected with the highest number of diseases (atramentous necrosis, pigmen-tation response, dark spots disease and coral hyperplasia). Site 6 has 7 diseases infecting 8 species, of which Porites solida is infected with the highest number of diseases (ulcerative white spots, black band disease, white plague and white patches). Sites 5 and 6, having low number and lowest percentage cover of coral diseases, are characterized by lowest levels of trace elements in both water and sediments. They are also characterized by nice and ideal slopes. Their coral diseases were reported only on the reef flat, probably because the reef flat is sheltered, lying below high mountains, a condition that may promote bacterial growth on the reef flat.

CONCLUSIONS AND RECOMMENDATIONS

Sedimentation may increase the percent coral diseases at the expense of disease number. However, the highest

percentage cover of algae/sea grasses is associated with the highest percentage cover of coral diseases. The coral disease atramentous necrosis attained the highest percentage cover in all sites which lie all in wadis being liable to terrestrial runoff. There is an evidence that vermetidae predation could be a causative agents of ulcerative white spots but Tridacna spp. could take the disease as a suitable substrate. The coral disease tissue coral neoplasia, being found only in site 4, was associated with high sediment load, low salinity due to fresh water coming from the adjacent tourist showers which are just close to the shore. Site 1 has 9 diseases infecting 12 coral species, of which Cyphastrea serialia is infected with highest number of diseases (atramentous necrosis, dark spots disease, skeletal eroding band and white plague). This site is characterized by the maximum metal concentrations of Zn, Cd, Pb and Ni in water and highest metal concentrations for Cu, Zn and Pb in sediments. Site 2 has 9 diseases infecting 10 corals, of which Millepora dichotoma is infected with the highest number of diseases (white plague, atramentous necrosis, brown band disease and coral hyperplasia). This site is characterized by the highest Cu concentration in water. The fewer number of coral diseases in site 4 attained the highest percentage disease cover indicating space monopolization of those diseases. This site attaind the the highest levels of Cd and Ni in sediments and highest percentage sediments. Sites 5 and 6, having low number and lowest percentage cover of coral diseases, are characterized by the lowest levels of trace elements in both water and sediments.

Identifying knowledge gaps that impede understanding coral disease mechanisms, and limiting elucidation of causes, significance or control of coral disease. Recommending directed research and education to fill these knowledge gaps. Standardizing methods for investigating coral disease outbreaks considering both biotic and abiotic etiologies. Addressing issues relative to the management of coral reef resources; and fosters collaboration among partners, stakeholders, key marine resource management agencies, and regional networks. Developing guidance for the proper handling and containment of corals in infectious disease experiments. Fostering the development of a cohesive coral disease research community.

ACKNOWLEDGEMENTS

This work was done in the frame of the strategy of the National Institute of Oceanography and Fisheries "Coral disease distribution as biomonitors of environmental impacts" for the year 2011/2012.

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Effect of ectomycorrhizae on growth and establishment of sal (Shorea robusta) seedlings in central India

ABHISHEK PYASI, KRISHNA KANT SONI, RAM KEERTI VERMA♥ Forest Pathology Division, Tropical Forest Research Institute, Post-Regional Forest Research Centre, Mandla Road, Jabalpur 482 021,

Madhya Pradesh, India. Tel.: +91-761- 4044003, Fax.: +91-761-2840484, 4044002, ♥e-mail: [email protected]

Manuscript received: 26 March 2013. Revision accepted: 7 May 2013.

Abstract. Pyasi A, Soni KK, Verma RK. 2013. Effect of ectomycorrhizae on growth and establishment of sal (Shorea robusta) seedlings in central India. Nusantara Bioscience 5: 44-49. The aim of the present study was to develop ectomycorrhiza in sal sapling at outside the sal growing areas. For this purpose sal seedling were raised at Jabalpur which is around 80 km away from natural sal forest (Motinala, MP). Seed sowing was done with inoculation of ectomycorrhizal inocula prepared by isolating the fungi from surface sterilised young basidiocarp of Lycoperdon compactum and Russula michiganensis. The inocula of ectomycorrhizal fungus were prepared in wheat grains treated with gypsum. The synthesis of ectomycorrhiza was observed in the sapling planted in the experimental field at Jabalpur with production of basidiocarp of Lycoperdon compactum near saplings. The mycorrhized saplings also showed higher growth indices.

Key words: ectomycorrhizal inoculum, ectomycorrhiza synthesis, nutrient uptake, sal forest

Abstrak. Pyasi A, Soni KK, Verma RK. 2013. Pengaruh ektomikoriza terhadap pertumbuhan dan pembentukan bibit sal (Shorea robusta) di India tengah. Nusantara Bioscience 5: 44-49. Tujuan penelitian ini adalah untuk mengembangkan ektomikoriza pada anakan pohon sal yang ditanam di luar wilayah pertumbuhan aslinya. Untuk itu bibit sal ditanam di Jabalpur yaitu sekitar 80 km jauhnya dari hutan alam sal (Motinala, MP). Penaburan benih dilakukan dengan inokulasi inokulum ektomikoriza yang disiapkan dengan mengisolasi basidiocarp muda jamur Lycoperdon compactum dan Russula michiganensis yang permukaannya disterilkan. Inokulum jamur ektomikoriza dipersiapkan dalam biji gandum yang diperlakukan dengan gipsum. Sintesis ektomikoriza teramati pada anakan pohon yang ditanam di kebun percobaan di Jabalpur dengan produksi basidiocarp Lycoperdon compactum di dekat anakan pohon. Anakan yang bermikoriza juga menunjukkan indeks pertumbuhan yang lebih tinggi.

Kata kunci: inokulum ektomikoriza, sintesis ektomikoriza, serapan hara, hutan sal

INTRODUCTION

Sal (Shorea robusta C.F. Gaertn.; Figure 1) is one of the most important sources of hardwood timber in India. Density of sal forest has significantly reduced from sal dense 65.6% in 1976 to 11.1% in the year 1999 followed by sal open 11.2% and sal medium 18.2%. The overall change has been estimated to be 42.1% of the total forested area (Chauhan et al. 2003). Decreasing sal forest cover is one of the top ranked problems of forest department. The primary reason for decreasing it so rapidly is poor regeneration. The seed of sal is recalcitrant, and start to germinate just before it detaches from the tree. It immediately needs appropriate moisture, nutrient and mycorrhiza for its establishment. A mycorrhiza in general is a symbiotic relation between a fungus and the roots of a vascular plant. Ectomycorrhization refers to the infestation of cortical tissues of root by hyphae of mycorrhizal fungi (Harley

Figure 1. Flowers of sal (Shorea robusta C.F. Gaertn.)

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1959). Mycorthe roots of mprovides the access to carsupplied by thfrom their soureturn, the platerms of wateplant's mineramay be incapdemineralisedmycelium of tthese phosphoplants they comycorrhizal fsuccession or found to have high metal cosoils and coal

Ectomycorecosystem as iof basidiomymain ectomycalready been fungi are: Astrfallax, GeasScleroderma Scleroderma cinerella, Rusmichiganensis2011, 2012). Tproduced on growing basidnutrients mainThe life of basproduction camonths.

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5 (1): 44-49, May 2013

46

For preparation of Ectomycorrhizal inocula the culture isolated as above were multiplied in water soaked boiled wheat grains treated with gypsum salt and a pinch of Bavistin in a narrow mouth glass bottles (Figure 3F). Six to eight mycelial agar discs of mycorrhizal fungi were inoculated in to the wheat grains. After 10-12 weeks of incubation at 25-270C the inoculum was ready for use (Kanan and Natrajan 1988).

Inoculation and planting of seedlings The experimental seedlings received the following

treatment and are arranged in CRD on a cemented platform, i.e. (i) Control, (ii) Sal litter, (iii) ECM1 (Russula michiganensis), (iv) Sal litter+ECM1, (v) Vermicompost+ RCM1, (vi) ECM2 (Lycoperdon compactum), (vii) Sal litter+ECM2, (viii) Vermicompost+ECM2, and (ix) Vermocompost.

Sal litter collected from sal forest of Motinala, Balaghat District, Madhya Pradesh, India (Figure 2) and added to the local soil in the ratio of 1:4 (v/v). The vermicompost was also mixed in the same ratio. The potting mix was filled in black polyethylene bags of 7x9". Ten g inocula of ectomycorrhizal fungi were given to each seedling of treatment numbers 3-8. The inoculum was placed just below seeds during seed sowing in pre monsoonal period. After five months the seedlings were planted in the micro-plots with 1x0.75m spacing at the Institute campus in RCBD. Four blocks each with all the above mentioned treatments were made. The seedlings were watered with tube well water as and when required.

Observation and recording of data The basidiocarp of ectomycorrhizal fungi are produced

on soil surface near inoculated seedlings. The life of basidiocarp was recorded 2-3 days but it production persist for more than 3 consecutive months of rainy season. Basidiocarp of Lycoperdon compactum are produced in close vicinity of roots in artificially inoculated sal saplings during July-August.

Heights of saplings, leaf breath and length were measured using standard centimetre scale. Diameter of saplings at collar region was measured using Varner's callipers. Leaf area index was calculated by multiplying maximum length of leaf with breadth (cm2). Growth indices of seedlings were calculated after 9 months of planting using the following formula:

HxCDxLAI Growth Indices = ---------------------- H+CD+LAI H = Height; CD = Collar diameter; LAI = Leaf area index

Statistical analysis Data obtained on growth indices were analysed by one

way analysis of variance (ANOVA) using Sx Statistics software.


Results Basidiocarps of Lycoperdon compactum were observed

in groups of 1-3 near ECM2 treated saplings in two blocks in close vicinity forming ectomycorrhizal association with feeder roots of sal at Jabalpur (Figure 3A-B). Mycorrhiza developed by the winter season (Figure 4A-B). No basidiocarp was observed in control and other treatments. Basidiocarps produced are short lived usually 2-3 days which upon maturation releases basidiospores. At Jabalpur basidiocarp was produced 3 times during July-August 2012 when rainy season was at its peak. The basidiocarp is rough, tough, globose, yellowish pinkish brown 2-5 cm in diameter, a typical gasteromyceteous shape, with a small rhizoidal stalk at base. Young sporophore is hard, depressed above, exoperidium glabrous, smooth 1-1.5 mm thick leathery, endoperidium smooth very thin. Gleba olivaceous, amber when young, becomes powdery chocolate brown at maturity. Spores abundant, round, hyaline to olivaceous brown, verrucose 4.5-5.0 µm (Figure 3C). The fungus grows on PDA agar medium with milky white, irregular colonies, rhizoidal, radiate, slow growing 35 mm. in diam after 7 days at 25±20C.

The maximum growth indices was observed in vermicompost+ECM2 treatment, which was 3.4 times more than control followed by ECM2, 3.0 times more, vermicompost+ECM1, 2.1 times more, sal litter+ECM2, 2.1 times more and sal litter+ECM1, 1.8 times more. Other treatments have no significant effect on growth indices of sal saplings (Table 1).

Table 1. Growth indices of sal saplings treated with different organic amendments and cultures of ectomycorrhizal fungi after 9 months of planting in micro-plots.

Cont

rol

ECM

1

Sal L

itter

Sal l

itter

+ECM

1

Verm

icom

post+

ECM

1

ECM

2 Sa

l Litt

er+E

CM2

Verm

icom

post+

ECM

2

Verm

icom

post

Growth indices

2.35 3.64 3.44 4.20 5.03 6.99 4.90 7.98 3.03 CD0.05 = 1.55 Note: ECM1 = Ectomycorrhizal fungus, Russula michiganensis; ECM2 = Ectomycorrhizal fungus, Lycoperdon compactum.

Discussion Mycorrhiza is a beneficial association between fungi

and root of higher plants. About 85 percent higher plants (gymnosperms and angiosperms) form this type of relationship in forests (Bakshi 1974). In fact this sustainable relationship is a deal between mycobionts and phycobionts for their survival and to some extent it may prove to be an obligatory for both the partners. This

PYASI et al. – Effect of ectomycorrhizae on Shorea robusta seedlings

47

Figure 3. Lycoperdon compactum, an ectomycorrhiza forming fungus associated with sal saplings. A. Fruit body near the sapling of Shorea robusta, B. Single basidiocarp, C. Basidiospores (40x), D. Seven days old pure culture on PDA plate. E. Seven days old pure culture on PDA slants, F. 45 days old spawn in wheat grains.

A A B

C D

E F

5 (1): 44-49, May 2013

48

Figure 4. Development of ectomycorrhiza in sal. A. Ectomycorhizal roots collected during winter season. B. Mantle formation in sal root beneficial relationship can be exploited in establishing new plantations at deteriorating sites and in degenerating forest covers. In the present study we established an ectomycorrhizal synthesis in vivo by inoculating two ectomycorrhizal fungi during seed sowing along with other treatment like sal litter and vermicompost which provides sal seeds the natural consortium of microbes which support its growth. But these natural consortiums of microbes when used alone are not very much effective in growth of sal saplings. These are more effective when used along with the cultures of ectomycorrhizal fungi. Among the two ectomycorrhizal fungi used only Lycoperdon compactum produces basidiocarps in short duration. Another species Lycoperdon perlatum and Russula parasurea are reported to form mycorrhiza with P. patula in Nilgiri Biosphere Reserve Tamil Nadu, India (Mohan 1991). In Malaysia two another dipterocarps also developed ectomycorrhiza with locally isolated ectomycorrhizal fungi (Lee et al. 2008). Many scientists from India and abroad have artificially established mycorrhization in Pinus and Eucalyptus spp. and have got fruitful results (Alexander 1981; Marx and Ross 1970). In central India, Sharma et al. (2009) have synthesise ectomycorrhiza in a monocot host, Dedrocalamus strictus with Cantharellus tropicalis. In our study we used wheat grains for production of inocula of ectomycorrhizal fungi similarly Kannan and Natrajan (1988) also produced spawn of Laccaria laccata and Amanita muscaria but in sorghum grains. In the present study we have tried sal litter collected from natural sal forest to inoculate the seedling in non-sal area similarly Lakhanpal (1987) used natural roots of mycorrhizal trees for artificial inoculation of Pinus gregardiana and Picea smithiana seedlings to develop mycorrhiza.

CONCLUSION

Artificial application of ectomycorrhizal fungus synthesise mycorrhiza in feeder roots at non sal growing area and helpful in initial growth and establishment of sal seedling in central India.

ACKNOWLEDGEMENTS

Authors are thankful to Dr. U. Prakasham IFS, Director Tropical Forest Research Institute (TFRI), Jabalpur, Madhya Pradesh, India for providing necessary facilities during course of study and Indian Council of Forestry Research and Education (ICFRE) Dehradun, India for financial assistance as Senior Research Fellowship under the project ID 136/TFRI/2009/Path-1 (15).

REFERENCES

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