R. T. Luzaran1 F.M. Dela Cueva2

1Philippine Sugar Research Institute, Victorias Expt’l Station, VMC Compound, Victorias City, Negros Occidental. Philippines

2Institute of Plant Breeding, University of the Philippines, Los Baños, College, Laguna. Philippines

*A technical paper presented at Asian Conference in Plant Pathology(ACPP) held in Darwin, Australia on April 27-29, 2011.

*

OV

ER

VIE

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characterized by the production ofunbranched whip like structure from the top ofinfected stalk

composed of host tissues and of fungus tissue

emerged at 2-4 months up to 6 – 7 months

developed systemically throughout the stalk

caused by the basidiomycetes fungus

Ustilago scitaminea Sydow.

Sugarcane Smut

through the bud

Teliospores germinate to produce the infectious, dikaryotic hyphae that penetrate the basal portion of young inner bud scales and invade the meristematic region of the bud.

smut infection can also take place in the soil when teliospores come in contact with germinating buds on seed setts.

Method of Infection

wind-borne spores

infected sugarcane cuttings (setts)

soil-borne inoculum

insect, animals, human beings and machinery

Mode of Spread

Imperata cylindrica

Sorghum vulgare

Roettboellia exaltata

Saccharum spontaneum

Alternative Hosts:

B(I) A (T)

T(I)Ta(T)

use of resistant cultivars

use of healthy and diseased free seed cane (setts)

roguing

heat therapy

non ratooning of heavily infested fields

Prevention and Control

To identify and characterize U. scitaminea isolates collected from different areas in the Philippines by molecular and phenotypic approaches.

1) to assess the genetic diversity among isolates ofthe sugarcane smut fungus using microsatellitemarkers,

2) to analyze the variation in pathogenicity (or theirability to produce disease) as well as morphologicalcharacters of the different U. scitaminea isolates,

3) to obtain information on the possible occurrenceof pathogenic races of smut fungus, if any in thePhilippines.

General Research Objective:

Specific Research Objectives:

The research was conducted at the Instituteof Plant Breeding (IPB), University of thePhilippines, Los Baños, College, Lagunaand at CIRAD (’Centre de cooperationinternationale en recherché agronomiquepour le development ‘) laboratory,Montpellier, France.

Fig 2.Neighbor joining tree of the 79 U. scitaminea genotypes isolated from 77 distinct sori collected in 15 different countries.

very low genetic diversity within aworldwide collection of smut ( Ustilagoscitaminea) exist in America and Africabut a lot is present in Asia in particularin the Philippines.

Smut whip collection

Fungal isolation

Mycelial Production

DNA extraction, quantification

PCR Amplification and Electrophoresis, Microsatellite Analysis

Pathogenicity Test

How it is done?:

TARLAC

VICTORIAS

PASSI

BUKIDNON

BATANGAS

SAGAY

PAMPANGA

CAPIZ

HPCO

LA CARLOTA

SONEDCO

ORMOC

BOGO

SAN CARLOS

LOPEZCADIZ

DAVAO

COTABATO

Figure 2. Map of the Philippine showing the collection sites of smut whips

Map taken from HPV

Mill No. ofDistrict samples

Luzon 25

Visayas 51

Mindanao 20

Total 96

Fig. 5. White mycelial colonies of U. scitaminea 3-5 days after spore plating.

Fig.6. Single sporidial isolates of U. scitaminea in PDA medium.

Collection and Isolation of U.scitaminea

Sampling sites:

RESULTS

Figure 7a. Isolated genomic DNA of various U. scitamineaisolates

Lane 1) Lambda 5 ng/ul, 2) Lambda 10 ng/ul, 3) Lambda 40ng/ul, 4) Lambda 50 ng/ul; 5) Isol. 21; 6) Isol. 22; 7) Isol. 24;8) Isol. 24; 9) Isol. 25; 10) Isol. 26; 11) Isol.43; 12) Isol. 53;13) blank (buffer) 14) Isol. 56; 15) Isol. 61; 16) Isol. 64; 17)Isol. 66; 18) Isol. 72; 19) Isol. 76; 20) Isol. 78; 21) Isol. 79;22) Isol. 80; 23) Isol. 82; 24) Isol. 83; 25) Isol. 91; 26) Isol.93; 27) Isol. 96.

1 2 3 4 5 6 7 8 9 1 0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Sample - 96 isolatesDNA - extracted from dried mycelium

DNA conc.- 5-50 ng/ul

Genetic Diversity of U. scitaminea

DNA quantification

* Diverse banding pattern in the gel indicated ahigh level of genetic variability within smut isolates

Figure 8a. Microsatellite profiles generated from genomic DNA of U. scitaminea isolates using USc 21 primer.

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dd

er(b

p)

•59

•60

•61

•62

•63

•77

•78

•79

•80

•81

•82

•83

•84

•85

•86

•87

•88

•89

•90

•91

•92

•93

•94

•95

•96

•La

dder

(b

p)•

41a

•90

a•

76a

•91

a•

87C

•94

b•

88a

•99

a•

Ladd

er

(bp)

300

290

280

270

260

250

240

230

DNA Isolates Control

Fragments of Band Scoring

Group A

Group B

Group C.

Figure 9. Neighbour joining tree of the 90 U. scitamineagenotypes collected from 17 different sugarcane districts in the Philippines. (Darwin’s software). Bootstrap values are at each node.

Cluster Analysis

no geographic patternwas evident becauseisolates from eachgeographic regions werespread throughout eachcluster.

Group B

Group A

Group C

Axis 2(22.53%)

Axis 1 (28.77%)

Figure 10. Plot defined by first two principalcoordinate analysis (PCoA) factors based ongenetic dissimilarity (simple matching coefficients)of 90 individual isolates of U. scitaminea. The firstand the second axes represent 28.77 and 22.53%of the variation respectively.

Principal Component Analysis (PCoA)

1) Isolate 2 (Batangas)

2) Isolate 20 (Tarlac)

3) Isolate 43 (San Carlos)

4) Isolate 76 (Cadiz)

5) Isolate 90 (Ormoc)

6) Isolate 61 (Bogo)

7) Isolate 88 (Bukidnon)

8) Isolate 57 (Davao).

Figure 11. Two month old experimental cane in screened mesh inoculated with U. scitaminea.

Front view

VARIATION IN PATHOGENICITY of U. scitamineaISOLATES

Preparation of inoculum and inoculation of the seedpieces

Table 2. Sugarcane varieties with known reaction to smut and parentage used as differential hosts. ________________________________________________ Varieties Parentage Reaction to Smut _____________________________________________________

VMC 86-5501 Phil 56-226 x ? SusceptibleNCo 3102 Co 421 x Co 312 SusceptibleVMC84-947 (PS 1)1 POJ2725 x ? ResistantF1343 Co290 x POJ 2878 IntermediatePS5 (V95-09)1 90 x ? Resistant_____________________________________________________Country of origin: 1 Philippines; 2. Natal, South Africa; 3 Formosa, Taiwan

* fresh population of teliospores * seedpiece inoculation

Pathogenicity Test

* 8 representative isolates

Number of infected plants% infection = ----------------------------- x 100

Total no. of plants

Percentage data= transformed to arc sine angles

Aggressiveness TestAggressiveness - average length of the incubation period of the disease on the test varieties.

Isolates which caused the test varieties to produce smutwhip infection within a shortest period of time wereconsidered aggressive and those isolates producing smutinfection with longest incubation period were consideredleast aggressive

Virulence of the Isolates

Anova of infected plants transformed to arc sine showed highly significant (P<0.01) effect of variety, isolate and var x isolate

differential varieties responded differently to the isolates. - magnitude of differences between isolates differed from variety to another.

0102030405060708090

Isolates

Perc

ent I

nfec

tion

(%)

VMC 86-550 Nco 310 VMC 84-947 F134 V95-09

Figure 12. Percent infection of five differential varieties to 8 isolates of U. scitaminea after 5 months of inoculation.

Virulence of the isolates on differential sugarcane varieties

Cdiz 80 & 59 % on F134/947,Dvo &Bats 2.5 & 5.62 947 & Nco

Fig.13. Black, whiplike sorusarising from the terminal meristem of a stalk infected byU. scitaminea.

Fig. 14. Side shoots on the sugarcane stalksbearing whip.

Fig. 15. Thin and grassy shoot appearance of inocu-lated susceptible variety, VMC 86-550.

Fig. 16. Emerging young shoots are more spindly and erect.

Fig. 17. Affected plant tillers profusely with small narrow leaves.

Characteristic symptoms of sugarcane smut

020406080

100120140

Batang

as

Tarlac

San C

arlos

Cadiz

Ormoc

BogoBuk

idnon

Davao

Isolates

Incu

batio

n pe

riod

(day

s)

VMC 86-550 Nco 310 VMC 84-947 F134 V95-09

Figure 18. Incubation period of eight isolates of U.scitaminea Syd. on five differential varieties.

The isolates varied significantly in aggressiveness as indicated by the time required for appearance of smut whip.

There were significant (P<0.01) effect on variety, isolates and their interaction on aggressiveness.

Comparison of aggressiveness of eight isolates of U. scitaminea

SC then Cadiz w/47 & 56 then Tarlac 80 days

Percent Infection Disease Reaction0-14% Resistant (R)15-23% Intermediate (I)24-60% and above Susceptible (S)

Design: CRD

Replications: 4 ( 10 plants/replication /variety)

Duration: 5 months

Recording of data as simple percentage of infected (presence or absence of a whip) plants within each clone.

Evaluation of Host Reaction

Table 8. Reaction of five differential sugarcane varieties to eight isolates of U. scitaminea Syd.

_________________________________________________________Isolates VMC NCo 310 VMC F134 PS-5

86-550 84-947 (VMC95-09) (S)* (S)* (R)* (I)* (R)*

________________________________________________________________1) Isolate 2 (Batangas) R R R S S 2) Isolate 20 (Tarlac) S R R S S3) Isolate 43 (San Carlos) S S I S I4) Isolate 76 (Cadiz) S S I S S5) Isolate 90 (Ormoc) I S I S S6) Isolate 61 (Bogo) S S I S S 7) Isolate 88 (Bukidnon) S S I I S8) Isolate 57 (Davao) S S R S S_______________________________________________________________*, known reaction to sugarcane smut

* There were seven distinct group of isolates producing distinct reaction types on five differential varieties

Evaluation of Host Reaction

Gross morphology of the isolates

Figure 19. Growth characteristic of eight representative U. scitamineaisolates in PDA medium 15 days after inoculation.

Isolates

1 2 3 4 5 6 7 8Rep 1

Rep II

RepIII

Morphological Characterization and Microscopic Examination of Different U. scitaminea isolates

Spore morphology and growth in culture did not vary much among the different isolates.

Table 10. Growth characteristics of eight representative isolates of U.scitaminea inPDA medium 15 days after incubation________________________________________________________________________

Colony Characteristics Isolate Colony color Mean colony diameter (mm) 1

__________________________________________________________________1) Isolate 2 (Batangas) Whitish brown 32.0 ns2) Isolate 20 (Tarlac) Cream white 31.53) Isolate 43 (San Carlos) Brown black 31.54) Isolate 76 (Cadiz) White mycelia 30.05) Isolate 90 (Ormoc) Brown black 32.56) Isolate 61 (Bogo) Brown black 31.37) Isolate 88 (Bukidnon) Cream to white 29.88) Isolate 57 (Davao) Brown black 32.3________________________________________________________________________Cv= 6.211 Observations were done on 15 plates per isolates

Figure 20. Teliospores of Ustilago scitaminea Syd (40X)

Figure 21. Sporidia of Ustilago scitaminea Syd (40X)

Gross morphology of the isolates

Table 11. Measurement of teliospores and sporidia of eight representative isolates and their color and wall markings characteristics.

_________________________________________________________________________________________Isolates Ave. teliospore Teliospore Ave thickness Wall Size of sporodia

diameter* color of spore wall* markings Length* Width*(µm) (µm) (µm) (µm)

___________________________________________________________________________________Isola 2 (Batangas) 7.05a brown 0.71cd echinulate 11.83a 2.22nsIsol. 20 (Tarlac) 6.77b brown 0.73c echinulate 10.82b 2.01Isol. 43 (San Carlos) 6.80b brown 0.71cd echinulate 11.33ab 2.27Isol. 76 (Cadiz) 6.50c light brown 0.85b echinulate 11.33ab 2.22Isol. 90 (Ormoc) 6.92ab brown 0.69d echinulate 10.72b 2.26Isol. 61 (Bogo) 6.55c brown 0.70d echinulate 11.80a 2.02Isol 88 (Bukidnon) 6.50c light brown 0.91a echinulate 11.80a 2.29Isol. 57 (Davao) 6.32d brown 0.84b echinulate 11.92a 2.12_________________________________________________________________________cv = 1.53 2.17 4.78 6.21* Average of 50 teliospores and 50 sporidia per isolate

Gross morphology of the isolates

No appreciable variation in terms of morphological characteristics of the smut isolates

Typical symptoms of sugarcane smut were produced byall isolates but it showed differences in virulence andaggresiveness when inoculated to five differential hosts

Frequent exchange or movement of sugarcane varietiesis likely the explanation for a significant variation amongU. scitaminea populations

Smut isolates in the Philippines are highly diverse interms of genetic and pathogenic characteristics

No appreciable variation in either spore size, color andwall markings were observed

Seven distinct group of isolates producing distinctreaction types on the five differential varieties were notedindicating that race of the fungus probably existed.

Research highlights:

The results revealed that U. scitaminea isolates inthe Philippines are highly diverse in terms of geneticand pathogenic characteristics. It is thereforerecommended that:

a) evaluation of breeding lines for resistance to sugarcanebe done in areas where isolates are more virulent andaggressive. e.q, Bogo, Medellin and / or Lilia, Cadiz City toensure high disease pressure and provide reliable reactionof materials for the disease.

b) knowing the existence of high genotype x isolatesinteraction, breeding works should aim at developing/releasing varieties that are location specific.

RECOMMENDATIONS

Acknowledgement:

PHILIPPINE SUGAR RESEARCH INSTITUTE, FOUNDATION, INC

INSTITUTE OF PLANT BREEDING, UP LOS BANOS, COLLEGE,

’Centre de cooperation internationale en recherché agronomique pour le development ‘) CIRAD, Laboratory, Montpellier, France.