i
SUSCEPTIBILITY AND TOLERANCE IN RED SKINNED POTATO
(SOLANUM TUBEROSUM L.) TO ROOT-KNOT NEMATODE,
MELOIDOGYNE SPP.
A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE
UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
TROPICAL PLANT PATHOLOGY
MAY 2012
By
Basil H. Kandouh
Thesis committee:
Brent S. Sipes, Chairperson
Koon-Hui Wang
Kent Kobayashi
ii
ABSTRACT
Susceptibility and tolerance to Meloidogyne, root-knot nematode (RKN), can be
different among potato cultivars. These differences extend to different species of RKN
within a potato cultivar. Most red skinned potato cultivars have not been studied in terms
of susceptibility and tolerance to Meloidogyne spp. This thesis examined susceptibility
and tolerance of eight red skinned potato cultivars to M. incognita at 4 population
densities in a 6-liter pot in Hawaii. Damage threshold of M. incognita was determined in
cultivars Desiree, Mountain Rose, Pink Pearl and Red Thumb. In the second experiment,
susceptibility and tolerance to M. javanica and M. konaensis were determined forthese
cultivars. Penetration and development rate of M. incognita and M. javanica were
compared in Mountain Rose and Red Thumb. At Pi 20, 200, or 2,000 nematode
eggs/plant, cultivars ‘Red Thumb’, ‘Durango’, and ‘Colorado Rose’ were susceptible
and intolerant, while ‘Rote Ersting’ was resistance but intolerant to M. incognita.
Although ‘Desiree’ was susceptible to all RKN species tested, it was moderately tolerant
to both M. javanica and M. konaensis, despite intolerant to M. incognita. ‘Mountain
Rose’ was moderately resistant and tolerant to M. incognita, and resistant/tolerant to M.
konaensis. However ‘Mountain Rose’ was susceptible and intolerant to M. javanica. TW
of ‘Mountain Rose’ was reduced due to M. javanica infections compared to TW of
uninoculated plants (P≤ 0.05). ‘Pink Pearl’ was susceptible/intolerant to M. incognita, but
susceptible/tolerant to both M. javanica and M. konaensis. ‘Red Thumb’ was the most
susceptible/intolerant cultivar to M. incognita, and the most resistant/intolerant to both M.
javanica and M. konaensis, among all cultivars tested. This research provides important
information on red skinned potatoes for breeders to develop new cultivars and for
iii
growers in choosing cultivars for fields infested with Meloidogyne. Crop response
information to this nematode can be efficiently used to develop effective crop rotation in
order to control Meloidogyne spp. in the tropics and subtropics.
iv
ACKNOWLEDGMENT
I would like to thank my committee Drs. Sipes, Wang, and Kobayashi for their help,
comments and efforts given through my study course and for being good friends as well.
I especially extend my deep appreciation to my major adviser, Dr. Sipes, for his unfailing
help, guidance, and supervision through my research. His criticisms were always
constructive.
I am very thankful and grateful to Donna Meyer and Michael Young not only for the
technical assistance but also for their extreme help in my daily life.
Thanks are given to Drs. Jarjees, Hu, Alveraz, Uchida, and all members in Department of
Plant and Environmental Sciences for being always pleasant and helpful.
I would like to acknowledge the Ministry of Higher Education of Iraq for awarding me
this scholarship and the Iraqi cultural Office in Washington DC for assisting me in order
to obtain the M.Sc. in Plant Pathology from the University of Hawaii, U.S.A.
Finally, special thanks go to my parents, brothers, sisters, and especially to my wife
Zinah and daughter Fatimah for their encouragement, support and enduring patience.
v
TABLE OF CONTENTS
Abstract .......................................................................................................................... ii
Acknowledgment ............................................................................................................ iv
List of tables ................................................................................................................... vi
List of figures ................................................................................................................vii
List of abbreviations .................................................................................................... viii
Chapter 1. Introduction ....................................................................................................1
References ...................................................................................................................9
Chapter 2. Susceptibility of red potato cultivars to Meloidogyne incognita..................... 15
Introduction ............................................................................................................... 15
Materials and Methods ............................................................................................... 16
Results ....................................................................................................................... 19
Discussion.................................................................................................................. 19
References ................................................................................................................. 22
Chapter3. Damage potential of M. incognita on 4 red skinned potato cultivars ............... 26
Introduction ............................................................................................................... 26
Materials and Methods ............................................................................................... 27
Results ....................................................................................................................... 29
Discussion.................................................................................................................. 33
References ................................................................................................................. 38
Chapter 4. Host efficiency and host parasitism of Miloidogyne spp. on red skinned
potato …………….………………………………………………………..………...40
Introduction .............................................................................................................. 40
Materials and Methods .............................................................................................. 41
Susceptibility to Meloidogyne spp. ........................................................................ 41
Host parasitism ....................................................................................................... 43
Results ....................................................................................................................... 44
Susceptibility to Meloidogyne spp. ........................................................................ 44
Host parasitism ....................................................................................................... 46
Discussion.................................................................................................................. 50
References ................................................................................................................. 52
vi
LIST OF TABLES
Table Page
2.1 Reproduction factor (Rf) and rank order for Meloidogyne incognita at different
initial populations (Pi) on red skinned potato cultivars ......................................... 21
3.1 Effect of different initial population densities of M. incognita on tuber weight
(TW) of red skinned potato and nematode reproductive ratio (Rf). ......................... 32
vii
LIST OF FIGURES
Figure Page
1.1 Internal and external symptoms of root-knot nematode infected potato tuber.….…. 4
2.1 Tuber weight (TW) differences among red skinned potato cultivars inoculated
with Meloidogyne incognita at different densities (Pi) ………………….…..…… 20
3.1 Effect of Meloidogyne incognita population densities (Pi) on plant shoot
growth of red skinned potato cultivars 35 days after inoculation. ……...….……… 30
3.2 Damage thresholds for Meloidogyne incognita on red skinned potato cultivars. ….. 34
3.3 Effect of M. incognita on plant root system and tuber quality of cultivar ‘Red
Thumb’...…………………………………………………………………….……... 36
4.1 The effect of three RKN species on potato tuber weight (TW) of four red potato
cultivars......…………………………………………………………...……..…….. 45
4.2 Reproduction factor (Rf) among four red skinned potato cultivars inoculated
with 10,000 eggs of M. incocnita, M. javanica or M. konaensis.…….....……..……47
4.3 J2 penetration at 12 DPI of M. incognita and M. javanica inoculated to
‘Mountain Rose’ and ‘Red Thumb’ red skinned potato ………………….….……. 47
4.4 Development and gall size comparison between M. incognita and M. javanica
on cultivar ‘Mountain Rose’ at 12 DPI……….……….………………….………...48
4.5 Developmental stages of Meloidogyne incognita...................................................... 49
4.5 Development and gall size comparison between M. incognita and M. javanica
oncultivar Red Thumb at 12 DPI …………………………………………….……. 49
viii
LIST OF ABBREVIATIONS
DPI Day post-inoculation
g Gram
HR Hypersensitive reaction
J2 Second-stage juvenile
mg Milligram
ml Milliliter
PCN Potato cyst nematode
Pf Final population
Pi Initial population of inoculation
Rf Reproduction factor (Rf= Pf/Pi)
RKN Root-knot nematode
spp. Species (plural)
TQ Tuber quality
TW Tuber weight
1
CHAPTER 1. INTRODUCTION
Resistance to root-knot nematode (RKN) is found naturally in many cultivated
plants (Fassuliotis, 1976; McSorley et al., 1999). Most potato cultivars (Solanum
tuberosum L.) are susceptible to RKN (Suri and Jayasinghe, 2002), although some have
been identified with resistance to RKN (Janssen et al., 1995 and 1997; Brown et al.,
2009). Potato is the fourth most important economic crop in the world, following rice,
wheat, and corn (Anonymous, 2009). People worldwide depend on the starch in potato to
fill their daily needs for carbohydrate. Red skinned potato cultivars have high nutritional
value and are important in the fresh market (Pardo et al., 2000). Red potatoes are
commonly used for soups and salads because of their firmness and texture. Their natural
pigmentation makes red potatoes favorable among customers and chefs (McComber et
al., 1994). Red potato has a high fiber (~ 25%), vitamin (C ~ 45%), Iron (~ 15%) and
relatively protein (~3%) content (http://www.nal.usda.gov/fnic/foodcomp). An increase
in the consumption of red potato is encouraged because of its high anthocyanin content
and antioxidant components which may reduce chances of cancers (Brown, 2005; Wang
and Stoner, 2008).
Potato might have the richest genetic diversity among all cultivated plants (Quiros
et al., 1991). There are about 5,000 potato cultivars and 200 wild species of potato
worldwide. Most of them found in southern Peru where potato is originated (OIA, 1989).
Solanum tuberosum is the major cultivated species of potato which is a tetraploid with 48
chromosomes. Most modern cultivars are related to this species. Four diploid species
with 24 chromosomes, two triploids with 36 chromosomes, and one pentaploid with 60
2
chromosomes are found in Solanum (Raker and Spooner, 2002). Solanum tuberosum is
divided into two major subspecies, S. tuberosum Andean and S. tuberosum Chilean.
Andean varieties are adapted to short days and mountainous equatorial and tropical
regions. Chilean potato varieties are adapted to high latitudes and long days (Solis et al.,
2007). Potato genetics are derived from various resources including wild relatives, native
cultivated species, locally developed varieties, and hybrids of cultivated and wild plants.
These resources broadly exhibit important traits, such as resistance to diseases and pests,
nutrition value, taste, and adaptation to extreme climatic conditions. Efforts have been
made to collect and characterize these traits and support a gene bank
(www.potato2008.org). Most commercial potato cultivars have been developed through
cross breeding to achieve desired traits (Brown et al., 2009).
Each major group of pathogens causes at least 2-3 common economic diseases on
potato shoots, roots or tubers. Important diseases on potato can be caused by fungi,
bacteria, viruses, viroides, and nematodes (Agrios, 2005; Hooker, 1981). Potato can be
infected by nematodes such as Globodera spp., Meloidogyne spp., and Ditylenchus spp.
(Hooker, 1981; Noling, 2009). Infection by RKN not only reduces potato yield but also
decreases the market value of the tubers increasing probability of product rejection and
thus drastic yield losses. More than 50% of the annual crop loss in potato is caused by
Meloidogyne spp. (Bird and Kaloshian, 2003).
The genus Meloidogyne includes about 100 species. Some species are considered
globally important and can cause severe damage on economic crops (Perry and Moens,
2006). Meloidogyne incognita, M. javanica, M. arenarea, M. hapla, M. chitwoodi, and
3
M. falaxa are considered the most important RKN species on potato (Netscher, 1970;
Jatala and Bridge, 1990; Brodie et al., 1993; Molendijk and Mulder, 1996). Meloidogyne
is a sedentary endoparasitic nematode feeding inside root and tuber cells. An egg mass is
deposited outside the female body. Under suitable conditions, eggs hatch to give second-
stage juveniles (J2). J2s move in search of host plants to complete their life cycle. Once
the J2 reach the plant root, the J2 penetrate the nearest root tips in the elongation zone
(Lohar and Bird, 2003). The J2 establishes a feeding site primary in phloem and/or
adjacent parenchyma cells (Viglierchio, 1991). Five to six cells serve as a feeding site.
These cells gain a high metabolic activity after modified by secretions of the root-knot
nematode(Hussey, 1989). The cells undergo hypertrophy meaning undergoing mitotic
divisions without cytokinesis (or cell wall formation), and form a dense granular
multinuclear cytoplasmknown as “giant cell”. . These giant cells extend mostly into the
stele of plant tissues (Perry and Moens, 2006). Adjacent cells around the feeding site will
undergo hypertrophy (abnormal of cell enlargement) and hyperplasia (abnormal increase
of cells numbers) reactions in all directions, forming a root gall (Viglierchio, 1991).
Like most vegetable crops infected by RKN, potato quality and quantity may be
decreased (Fig. 1, Noling, 2009). The host-pathogen relationship differs from species to
species, and may vary due to climate change (Brodie et al., 1993). Direct yield loss
occurs when RKN infect plant roots and disrupt water and nutrition uptake, thus
disturbing plant photosynthesis (Bridge, 1992; Williamson and Hussey, 1996).
4
Figure 1. Internal and external symptoms of root-knot nematode infected potato tuber. A)
infected tuber (left) and non-infected tuber (right). B) A peeled white potato infected with
Meloidogyne incognita. C) Specks due to root-knot nematode infection on a red skinned
potato.
Several symptoms can be observed and related to nematode infection in potato.
Foliar parts of infected plants particularly show less thriftiness, premature wilting and
leaf chlorosis; leading to plant death or yield reduction. Indirect loss is caused by quality
reduction when the nematodes infect the potato tubers. Root galling can indicate and
confirm RKN presence, infection level and damage potential. Infected potato may show
5
outer deformation of the tuber surface such as galls and bumps. Inner infections cause
blemishes and specks (Chaves and Torres, 2001). When peeled, RKN infected potato
shows visible minute brown specks (Fig. 1 B and C). Each speck is a group of infected
cells surrounding an adult female and her maturing egg mass (Tordable et al., 2008).
Tuber quality for marketing is often more important than yield quantity aspects.
Damaged tubers with galls, specks, or other deformities can easily lead to product
rejection. An entire potato shipment can be rejected due to an infested tuber (Ingham et
al., 2007). Moreover, Meloidogyne spp. can disseminate from an infected area to non-
infected area by transportation of seed tubers (Vovlas et al., 2005), further compounding
problems across fields.
RKN is difficult to control due to its wide host range and complicated
reproductive behaviors. Most common nematode management methods include crop
rotation of poor or less susceptible crops, resistant varieties, cultural and tillage practices,
and pre-plant soil fumigation treatments (Noling, 2009; Ingham et al., 2007; Perry and
Moens, 2006). Cultural control and land management may or may not be an effective
approach based on soil type, moisture, temperature and other related factors. The farm
condition can be very important to determine certain cultural practices in order to achieve
effective nematode management. Using such practices can noticeably reduce nematode
population densities but not below economic damage levels, especially when the farm is
continuously planted to susceptible cultivars. Pesticides are necessary to improve crop
yield in many cases. Using unrelated crops in crop rotation programs can substantially
control several kinds of soil borne problems including nematodes. In the case of RKN
and potato, it is difficult to find a successful crop with which to rotate to. Moreover with
6
an infested farm, one or two season fallow cultivation with of cover crop is required for
effective in reduction of nematode population density. Wheat or other cereal crops can be
rotated with potato especially in warm climates (Prasad, 2008). Sorghum-sudangrass (a
silage crop) has been reported to suppress RKN populations when rotated with potato in
warmer climates (Kratochvil et al., 2004). Soil management practices can help to reduce
nematode populations (Noling, 2009). Immediately or as soon as possible after the crop,
land should be disked after harvesting to expose all possible host plant tissue to
desiccation and death. Nematode populations can be effectively reduced by disking twice
and incorporating a short fallow in order to expose more soil to desiccation and sunlight
(Noling, 2009).
Chemical control for RKN can be necessary to avoid qualitative yield loss. On
the other hand, using nematicides to control plant-parasitic nematodes has been limited
due to prohibition for concerns over the environment and health (Jeayratnam, 1990).
Besides environment concerns, nematicides are expensive (Ristaino and Thomas, 1997).
Nematicides used currently include two major forms; fumigants and non-fumigants.
Fumigant nematicides are phytotoxic and restricted to pre-plant application. Unlike
fumigants, non-fumigant nematicides are less broad spectrum and can be used as pre-
plant and/or post-plant treatments to control nematodes in potato fields. Except for
Vydate which can be applied to the soil or foliarly, all non-fumigant nematicides are soil
applied (Noling, 2009). Non-fumigants such as Vydate can reduce nematode invasion
and affect development of nematode juveniles (Wright et al., 1980). Non-fumigant
nematicides can be systemic and protect the plant after being up taken by the plant root
system (Ingham et al., 2007).
7
Practically, resistant cultivars provide the best economical nematode control.
Potato cultivars with resistance to Globodera (PCN) are more commercially available
than those with resistance to RKN. The PCN resistance gene confers a broad spectrum of
activity for almost all Globodera spp. and is found in a range of wild potato species
(Rouppe van der Voort et al, 1998). However there is only one gene, RMc1, that gives
resistance to M. chitwoodi found in the wild potato species S. bulbocastanum. RMc1,
located on chromosome XI, and was intergrated into cultivated potato by somatic
hybridization (Gebhardt and Valkonen, 2001). During the two last decades, RKN
resistant potato cultivars have been commercialized and distributed widely among
temperate zones, but not in tropical or subtropical areas (Brown et al., 2009). Potato
cultivars resistant to RKN are mostly developed by crossing high yield commercial
cultivars with resistant wild relatives (Watanabe et al., 1994). In most of these cases, heat
stable resistance along with commercial traits is difficult to achieve (Berthou et al.,
2000).
Potato cultivars can behave differently to challenge by Meloidogyne spp.
(Charchar, 2009; Brown et al., 2009). Many studies have been conducted to determine
nematode susceptibility in Russet and Idaho white potato cultivars but not on red skinned
potato cultivars. Scarcity of information on red skinned potato and its interaction with
root-knot nematode may lead to choosing a susceptible cultivar and that will lead to high
level of damage and yield loss. Moreover, cultivation of susceptible cultivars can increase
the nematode population such that the next crop will be seriously threatened and the field
highly infested (Vovlas et al., 2005). Thus more information on red skinned potatoes and
8
their behaviors in tropical and subtropical areas is needed. The susceptibility and
tolerance to the RKN Meloidogyne spp. among red skinned potato must be investigated.
In plant pathology, susceptibility and tolerance are two different terms used to
determine and describe the relationship between the host plant and pathogen. In plant
nematology, cultivars are susceptible to nematodes when the plant supports high levels of
nematode reproduction (Canto-Saenz, 1985; Williamson, 1998). Thus susceptibility can
be measured by the pathogen reproductive factor or the final population from an infected
host plant. Unlike susceptible cultivars, tolerant cultivars have less or no differences from
uninoculated plants in terms of growth or yield in the presence of nematodes (Cook,
1974). A tolerant cultivar can be either resistant or susceptible and vice versa with a
resistant or susceptible cultivar. A combination of both resistance and tolerance can be
measured and found in some economic crops including potato (Canto-Saenz and Brodie,
1986). Selecting potato cultivars resistant and tolerant to Meloidogyne spp. is an effective
approach to maintain high yield, mitigate yield costs and manage the nematode.
In this thesis we hypothesized that different levels of susceptibility and tolerance
to Meloidogyne spp. can be found in cultivated red skinned potatoes. Therefore a series of
experiments were conducted to i) investigate susceptibility in eight red skinned potato
cultivars to M. incognita, ii) determine the damage potential of M. incognita on 4 red
skinned potato cultivars, iii) compare the susceptibility of 4 red skinned potato cultivars
to M. incognita, M. javanica and M. konaensis, and iv) evaluate the penetration and
development rate of M. incognita, and M. javanica on 2 red skinned potato cultivars.
9
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Williamson, V.M. and Hussey, R. S. 1996. Nematode pathogenesis and resistance in
plants. The Plant Cell 8:1735-1745.
14
Wright, D.J., Blyth, A.R.K., and Pearson, P.E. 1980. Behaviour of the systemic
nematicide oxamyl in plants in relation to control of invasion and development of
Meloidogyne incognita. Annals of Applied Biology 96: 323–334.
15
CHAPTER 2. SUSCEPTIBILITY OF RED POTATO CULTIVARS TO
MELOIDOGYNE INCOGNITA
Introduction
Susceptibility and resistance are terms used to describe the relationship between
the plant and nematode (Perry and Moens, 2006). Susceptibility of a host is measured by
nematode reproduction rate. Host efficiency or ‘susceptible-resistant’ is a continuum
from low to high measurements. Plants including potato are susceptible when they allow
nematodes to reproduce to or above ‘substantial level’, usually when the final population
(Pf) is equal to or greater than the initial population (Pi). Plants that support no or very
low nematode reproduction are resistant. However, a plant can be resistant but either
possess tolerant or intolerant property. Likewise susceptible plants may be tolerant or
intolerant. Nematode tolerance is measured by the level of plant damage due to nematode
infection regardless of their susceptibility levels. Intolerant plants suffer severe damage
or even death due to nematode infection (Canto-Saenz and Brodie, 1984 and 1986).
Meloidogyne chitwoodi has been reported as a major pest on potatoes in the major
potato production regions of the United States, Colorado, Idaho, Utah, Washington,
Northwest California and southeast Oregon (Pinkerton and McIntyre, 1987; Nyczepir et
al., 1982; Ingham et al., 2007). The cool climate favorable for growing potato is also
favorable for M. chitwoodi which is adapted to a long warm growing season and cool soil
temperature (O’Bannon and Santo, 1984). Since Russet Burbank (an Idaho type potato)
and white potato are the most common cultivated potatoes in the main regions of potato
production, tremendous research on how to manage M. chitwoodi had been conducted.
16
For example, resistance to M. chitwoodi is found in diploid potato and has been
intergraded into the cultivated potato (Brown et al., 1997, 1999 and 2009). However, M.
incognita and M. javanica are major pests in the tropics and subtropics causing
substantial yield losses in most crops including potato (Noling 2009; Vovlas et al., 2005).
Unlike resistance to M. chitwoodi in temperate climate regions, a successful resistant
potato has not been found for tropical and subtropical climates yet. Strenuous efforts are
made not only to determine and transfer resistance genes from wild to cultivated potato
but also to overcome the lack of heat stability in such genes (Berthou et al., 1996 and
2003; Hartman et al., 1982).
Potato cultivars resistant to M. incognita can be determined based upon gall
indices, reproductive ratios, and or incompatible responses such as hypersensitive
reactions and nematode penetration ability (Dropkin, 1969; Sasser et al., 1984; Canto-
Saenz and Brodie, 1986). Reproductive ratios or reproductive factors (Rf) are the most
common and reliable criteria to determine host susceptibility, while the plant gain (potato
tuber weight) can efficiently indicate tolerance levels of the plants to the nematode
infection. The objective of this chapter is to screen a range of red skinned potato
cultivars, for their resistance and tolerance to M. incognita, a common RKN species in
the tropic climate of Hawaii.
Materials and Methods
Meloidogyne eggs used for inoculum were collected from tomato plants Solanum
lycopersicum cultivar Pixie, cultured originally with a single egg mass. Tomato roots
were gently washed with tap water and cut to 1-cm long pieces and placed in a 500 ml
flask. The flask was shaken for 4 minutes with 0.5% NaOCl using a wrist action shaker
17
(Hussey and Barker, 1973). The liquid was passed through a 100-µm pore sieve and
collected from 20-µm pore sieves after washing with tap water (McSorley and Parrado,
1981). The eggs were counted and adjusted to 1000 eggs/ml.
Certified seed potatoes of ‘All Red’, ‘Colorado Rose’, ‘Desiree’, ‘Durango’,
‘Mountain Rose’, ‘Pink Pearl’, ‘Red Thumb’ and ‘Rote Ersting’ were planted
individually in 6-liter clay pots filled with 2:1 sterile sand/soil. Pots were placed on
benches and maintained in a shade house. Irrigation was applied as needed. Pre-plant
pots were incorporated with 25g of 16-16-16 (All purpose Gaviota, BEI Hawaii)
fertilizer/pot and followed by three additional applications during the growing period.
One week after planting, the emerged plants were inoculated with different initial
population densities (Pi) of M. incognita at 0, 20, 200 or 2000 eggs/pot diluted in 10 ml
aliquots. Ten weeks after inoculation, potato tubers were collected and weighed.
Experimental units were arranged in randomized complete blocks with two replications.
At harvest, a 250 cm3
soil sample was taken from each pot for elutriation (Byrd et al.,
1976). Tubers from each pot were chopped into 1-cm2 pieces and mixed. A 30 g tuber
subsample from each pot was placed in the Baermann funnels in a mist chamber to
extract J2 (Seinhorst 1950; Southey, 1986). After 6 days of incubation, nematodes
collected from the Baermann funnel were centrifuged (Coolen and D’Herde, 1972;
McSorley et al., 1999) and passed through a 25-µm pore sieve to collect nematodes.
Another 60 g tuber subsample from each pot was subjected to NaOCl egg extraction
(Hussy and Barker, 1973). Nematodes from the three extraction methods were counted
with the aid of an inverted microscope, and combined to give the final nematode
18
population density (Pf). A reproduction factor (Rf) was calculated by dividing the Pf by
Pi (Rf= Pf/Pi).
Data were analyzed using one-way analysis of variance. Tuber weight (TW) was used
to indicate tolerance among cultivars while Rf was used to indicate susceptibility among
cultivars. Variances were evaluated and nematode data were transformed [log10 (X+1)]
adjust normality of the data distribution. Means of the two replications were calculated.
Data collected were subjected to non-parametric analysis where cultivars were ranked
based on their TW, and Rf for each Pi from the highest to lowest Rf and the means of
their rank were calculated. Ranks from first to eighth indicated Rf (s) from the highest to
lowest susceptibility. Cultivars with Rf > 2 were considered very susceptible, those with
2 > Rf ≥1 were susceptible, whereas cultivars with Rf < 1 were considered resistant.
Results
Tuber weight (TW) of ‘Desiree’, ‘Pink Pearl’ and ‘Mountain Rose’ was not
affected by all inoculation levels (Pi) tested compared to TW of uninoculated plants (Fig.
2.1). However, TW of Cultivars ‘Rote Ersting’, ‘All Red’ and ‘Colorado Rose’ was
affected by the nematode populations especially at Pi 2000, greater than 20% TW
reduction compared to TW of the control. ‘Red Thumb’ and ‘Durango’ had a moderate
TW reduction (>10%) at Pi 2000 compared to TW of control plants. No effect on TW of
all cultivars was detected at Pi 20 or Pi 200.
Rf differed among cultivars for each Pi (Table 2.1). At Pi 20, ‘Desiree’ had the
highest Rf followed by ‘Colorado Rose’ and ‘All Red’ respectively. Other cultivars had
almost the same Rf value. ‘Mountain Rose’ supported the least nematode reproduction.
‘Desiree’, ‘Pink Pearl’ and ‘Colorado Rose’ had the highest Rfs at Pi 200. ‘Mountain
19
Rose’ had the lowest Rf (Rf ≤ 0.5). At Pi 2000, ‘Red Thumb’ had the highest Rf followed
by ‘Pink Pearl’ and ‘Desiree’ respectively. ‘Mountain Rose’ had the lowest Rf (Rf ≤ 0.1).
Overall Rf ranking was average of ranking from all three Pis (Table 2.1) showed that
‘Desiree’, ‘Pink Pearl’ and ‘Red Thumb’ supported the most nematode reproduction (Rf
≥ 4). ‘Colorado Rose’ and ‘All Red’, ranks 4 and 5, were in the middle range for the
tested cultivars. ‘Durango’, ‘Rote Ersting’ and ‘Mountain Rose’ had Rf <1 signifying
resistance to M. incognita.
Discussion
Red skinned potato cultivars exhibited different levels of resistance to M.
incognita. Other researchers have discovered similar results on white and Idaho potatoes
generalized not only with species but also races of nematode within a species (Brown et
al., 2009). In this experiment, ‘Desiree’ and ‘Pink Pearl’ were the most susceptible-
tolerant cultivars. Although ‘Red Thumb’ ranked 5 in overall Rf, it was the highest
susceptible cultivar at Pi 2000 supporting the greatest nematode population (Rf ≥ 2). TW
of Red Thumb was greatly reduced due to M. incognita suggesting this cultivar is
susceptible-intolerant. Interestingly, ‘Mountain Rose’ was the only resistant cultivar (Rf
= 0.4) with lowest Rf among all cultivars tested. Since the M. incognita population was
reduced and had no effect on ‘Mountain Rose’ TW, this suggests possible resistant to M.
incognita with an acceptable level of tolerance.
20
Figure 2.1 Tuber weight (TW) differences among red skinned potato cultivars inoculated
with Meloidogyne incognita at different densities (Pi= initial inoculation number of
nematode eggs/plant). Letter(s) A or AA refers to TW reduction greater than 10% or 20%
respectively.
21
Table 2.1 Reproduction factor (Rf) and rank order for Meloidogyne incognita at different
initial populations (Pi) (20, 200, or 2000 eggs/plant) on red skinned potato cultivars. The
lowest rank number refers to the highest Rf.
Cultivar Pi 20 Pi 200 Pi 2000 Overall
Rf Rank Rf Rank Rf Rank Rank
All Red 1.3 3 0.5 4 1.5 5 4
Colorado Rose 1.7 2 3.2 3 2.5 4 3
Desiree 2.6 1 7.3 1 8.5 3 1
Durango 0.0 6 0.2 5 0.8 6 6
Mountain Rose 0.0 6 0.0 7 0.1 7 8
Pink Pearl 0.1 4 6.2 2 9.8 2 2
Rote Ersting 0.1 4 0.0 7 0.1 7 7
Red Thumb 0.0 6 0.1 6 13.9 1 5
22
It has been demonstrated that different parts of the same plant can have a different
response to the same nematode, i.e. roots may be resistant to a certain Meloidogyne sp.
but the tuber is susceptible, and this might be were resistant but intolerant (Charchar,
2009; Brown et al., 2009). In another word, cultivars may not support nematode
reproduction but TW may be reduced at least 10% or more due to M. incognita
infections. ‘Desiree’, an old and widely used cultivar, has a high degree of tolerance to
M. incognita. ‘Mountain Rose’ and ‘Pink Pearl’ are another two promising cultivars
tolerant to M. incognita. However, ‘Mauntain Rose’ has advantages over ‘Pink Pearl’ as
it is also resistant to M. incognita.
Choosing a resistant potato cultivar with an acceptable yield may be an efficient
technique for more effective crop rotation. The information from this research on red
skinned potato cultivars will be useful for breeders and growers in selecting cultivars
tolerant and resistant to M. incognita, and thus mitigating yield loss.
References
Berthou, F., Kouassi, A., Bossis, M., Dantec, J. P., Eddaoudi, M., Ferji, Z., Pellé, R.,
Taghzouti, M., Ellissèche, D., and Mugniéry, D. 2003. Enhancing the resistance of the
potato to southern root-knot nematodes by using Solanum sparsipilum germplasm.
Euphytica 132:57-65.
Berthou, F., P. Rousselle, and Mugniéry, D. 1996. Ex Solanum sparsipilum heat-stable
resistance to Meloidogyne arenaria in Solanum tuberosum. III International Congress of
Nematology. La Guadeloupe, Program and Abstract: 101.
Brown, C. R. 2005. Antioxidants in potato. American Journal of Potato Research 82:163-
172.
23
Brown, C. R., Mojtahedi, H., and Santo, G. S. 1999. Genetic analysis of resistance to
Meloidogyne chitwoodi introgressed from Solanum hougasii into cultivated potato.
Journal of Nematology 31:264–271.
Brown, C. R., Mojtahedi, H., Santo, G. S., and Williamson, V. M. 1997. The effect of the
Mi gene in tomato on reproductive factors of Meloidogyne chitwoodi and M. hapla.
Journal of Nematology 29:416–19
Brown, C. R., Mojtahedi H., Zhang, L. H., and Riga, E. 2009. Independent resistant
reactions expressed in root and tuber of potato breeding lines with introgressed resistance
to Meloidogyne chitwoodi. Phytopathology 99:1085-1089.
Byrd, D. W., Barker, K. R., Ferris, H., Nusbaum, C. J., Griffing, W. E., Small, R. H., and
Stone, C. A. 1976. Two semi-automatic elutriators for extracting nematodes and certain
fungi from soil. Journal of Nematology 8: 206-212.
Canto-Saenz, M. and Brodei, B. B. 1986. Host efficiency of potato to Meloidogyne
incognita and damage threshold densities on potato. Nematropica 16:109-161.
Canto-Sánez, M. and Brodie, B.B. 1984. The nature of potato incompatible response to
Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949 and correlation between
root and tuber resonse. Journal of Nematology 16: In press.
Chrachar, J. M. 2009. Penitration, development, and reproduction of Meloidogyne
incognita race 1 and M. javanica on potato cultivars in the field. Nematologia Brasileira
2: 147-153.
Coolen, W. A. and D’Herde, C. J. 1972. A method for the quantitative extraction of
nematodes from plant tissue. Ghent State Agricultural Research Centre, State
Entomology and Nematology Research Station, Merelbeke, Belgium, 36 pp.
Dropkin, V. H. 1969. The necrotic reaction of tomatoes and other hosts resistant to
Meloidogyne reversed by temperature. Phytopathology 59:1632-1637.
24
Hartman, K., Sasser, J. N., and Jatala, P. 1982. Evaluation of potato lines for resistance
to major species and races of root-knot nematodes (Meloidogyne species). In: W.J.
Hooker Ed. Research for the Potato in the Year 2000, pp. 94–95. International Potato
Centre, Lima.
Hussey, R. S. and Barker, K. R. 1973. A comparison of methods of collecting inocula of
Meloidogyne spp. including a new technique. Plant Disease Reporter 57:1025-1028.
Ingham, R. E., Hamm, P. B., Baunf, M., David, N. L., and Wade, N. M. 2007. Control
Meloidogyne chitwoodi in potato with shank-injected metam sodium and other
nematicides. Journal of Nematology 39:161-168.
McSorley, R., Frederick, J. J. and McGovern, R. J. 1999. Extraction of Meloidogyne
incognita from caladium corms. Nematropica 29:245-248.
McSorley, R. and Parrado, J. L. 1981. Effect of sieve size on nematode extraction
efficiency. Nematropica 11:165-174.
Noling, J. W. 2009. Nematode management in potatoes (Irish or White). Florida Institute
of Food and Agricultural Sciences, University of Florida.
Nyczepir, A.P., J.H. O'Bannon, G.S. Santo, and A.M. Finley. 1982. Incidence and
distinguishing characteristics of Meloidogyne chitwoodi and M. hapla in potato from the
northwestern United States. Journal of Nematology 14:347-353.
O’Bannon,J. H. and G. S. Santo. 1984. Effect of soil temperature on reproduction of
Meloidogyne chitwoodi and M. hapla alone and in combination on potato and M.
chitwoodi on rotation plants. Journal of Nematology 16:309-312.
Perry, R. N. and Moens M. 2006. Root-knot nematodes: Plant nematology. Wallingford,
UK. CABI. Pp. 30-90.
Pinkerton, J. N. and McIntyre, G. A. 1987. Occurrence of Meloidogyne chitwoodi in
potato fields in Colorado. Plant Disease 71:192.
25
Sasser, J. N., Carter, C. C., and Hartman, K. M. 1984. Standardization of host suitability
studies and reporting of resistance to root-knot nematodes. A cooperative publication of
the Department of Plant Pathology, North Carolina State University and the United States
Agency For International Development.
Seinhorst, J.W. 1950. De betekenis van de toestand van de grond voor het optreden van
aantasting door het stengelaaltje (Ditylenchus dipsaci (Kühn) Filipjev). Tijdschrift over
Plantenziekten 56:289–348.
Southey, J. F. ed. 1986. Laboratory methods for work with plant and soil nematodes.
HMSO Books, Norwich, UK.
Vovlas, N., Mifsud D., Landa B. B., and Castillo, P. 2005. Pathogenicity of the root-
knot nematode Meloidogyne javanica on potato. Plant Pathology 54:657- 664.
26
CHAPTER3. DAMAGE THRESHOLD OF M. INCOGNITA ON FOUR
RED SKINNED POTATO CULTIVARS
Introduction
Since resistant and tolerant cultivars are the most economic approach to manage
root-knot nematode (RKN), desired cultivars must be tested in order to determine their
damage threshold. Damage threshold is the certain population of a pathogen which can
cause a certain level of yield loss. Yield loss due to RKN varies based on cultivar plant
species, nematode species, race, soil type and the environment (Barker and Olthof, 1976).
In case of plant-parasitic nematode (PPN), damage threshold is mostly determined by
linear or exponential relationship estimating a 10% yield loss at an initial population
density (DiVito et al., 1985; Ferris et al., 1994; McSorley et al., 1992). Thus knowing at
what nematode population density yield loss occurs will aid in selecting the appropriate
cultivar as well as provide basic information to develop integrated pest management
strategies against RKN on potato. Yield loss on potatoes can be measured qualitatively
and quantitatively based on RKN species, RKN race, and RKN population density. Ferris
et al. (1994) found that less than 1 Meloidogyne chitwoodi/cm3 soil could cause 10%
blemish on potato and the estimated threshold damage is about 1 egg/250 cm3 (Santo et
al., 1981). However the damage threshold of M. hapla on potato is 50 eggs/250 cm3
soil
(Brodie et al., 1993), higher than that for M. chitwoodi but much lower than that for M.
javanica which is 125 eggs/cm3
soil (Vovlas et al., 2005). For M. incognita, damage
threshold density was different between closely related clones of potato (Canto-Saenz
and Brodie, 1986). Only 5 eggs/cm3
soil caused > 10% tuber weight reduction on
Solanum tuberosum ssp. andigena and 10 eggs/ cm3 for S. sparsipilum x (S. phureja x S.
27
tuberosum). Almost all the above researches on RKN threshold on potato are based on
white and yellow skinned potato or Idaho potato. However, information on resistance
and tolerance of red skinned potato cultivars to Meloidogyne spp. is lacking or not exist.
This has become a constraint to red skinned potato cultivation particularly in subtropical
climates. Thus, the objective of this thesis chapter is to determine host efficiency and
damage threshold of M. incognita on four red skinned potato cultivars selected based on
preliminary screening results from Chapter 2.
Materials and Methods
Nematode inoculum. Meloidogyne incognita eggs masses were maintained in the
green house individually in tomato (Solanum lycopersicon cultivar Pixie). Egg inocula
for all tests were collected from 3-month old infected tomato roots. Tomato roots were
gently washed free of soil, cut into 1-cm long pieces and placed in a 500-ml flask. The
flask was shaken for 4 minutes with 0.5% NaOCl using a wrist action shaker (Hussey and
Barker, 1973). The liquid was passed through a 100-µm pore sieve and collected from a
20-µm pore sieve after washing with tap water (McSorley and Parrado, 1981). The eggs
were counted and adjusted to 1000 eggs/ml.
Experimental design. Damage potential of M. incognita on four red skinned
potato cultivars was determined in a pot experiment in a shade house. This was a 44
factorial (potato cultivars nematode population densities) designed experiment,
arranged in randomized complete block design, with four replications. The experiment
was run twice.
28
Potato cultivars ‘Desiree’, ‘Mountain Rose’, ‘Pink Pearl’, and ‘Red Thumb’ were
selected for this experiment based on their difference in susceptibility to M. incognita
(Chapter 2). Certified seed potatoes of the four cultivars were planted individually in 6-
liter pots. The pots were filled with 2:1 sterile sand/soil for the first trial or 1:1:1 sterile
sand, soil, and BIG R (red wood soil conditioner, Kellogg Garden Products, Honolulu,
HI) for the second trial. Plants were hill with 2-3 cm of the same media 3 times
throughout the growing period. The pots were placed on benches and maintained under
the ambient conditions. Irrigation was applied as needed. Pre-plant fertilization was a
treatment of 16-16-16 (All purpose Gaviota) fertilizer at 25 g/pot, followed by three
additional 25 g/pot during the growing period. The first trial of the experiment was
conducted from June 10, 2011 to September 22, 2011, while the second trial was
conducted from December 5, 2011 to February 15, 2012. One week after planting, the
emerged plants were inoculated with the RKN eggs. The initial population (Pi) was 0, 20,
200 and 2000 M. incognita eggs/plant in the first trial, and 0, 500, 5000 or 10000 M.
incognita eggs/plant in the second trial. The Pi was increased in Trial 2 because the initial
Pi levels were found to be below the damage threshold in Trial 1.
Data collection and analysis. Plant height was recorded 35 days post inoculation
(DPI). The plants were grown for a total of 8-10 weeks based on its senesces time. As
each cultivar senesced, tubers were harvested, weighed, chopped into 1-cm2 pieces and
shaken along with the roots in 0.5% NaOCl for nematode egg extraction (Hussy and
Barker, 1973). The same extraction method was used with the roots of each plant. In the
second experiment, chopped tubers were blended with 0.5% NaOCl in a food processor
for 10 seconds on low speed, and then the liquid was poured in 500ml flasks and shaken
29
for 4 minutes. The eggs were collected on a 25µm pore sieve and counted. Tuber quality
(TQ) was rated using an infection index in a scale of 0-6, 0=no infection, 1= 1-3, 2=4-5,
3-6-9, 4=10+, 5=50+, and 6=100+ infection sites/tuber (Pinkerton and Santo, 1986).
Susceptibility among cultivars to M. incognita was compared based upon data of
Rf (Pf/Pi), TW, TQ, and plant height. Data were log10 (x + 1) transformed to normalize
the data before analysis (Noe, 1985), and Waller-Duncan’s multiple range test at P < 0.05
was used for means separation wherever appropriate. TW was regressed to Pi for each
cultivar. To determine nematode threshold for each cultivar, data from Trial 1 and Trial 2
were pooled together with data for experiment 2. Data were subjected to Proc. Reg. using
SAS (SAS, Inc., Cary, NC).
Results
‘Red Thumb’ plants showed severe stunting, wilting, and yellowing 35 DPI (Fig.
3. 1. A). ‘Red Thumb’ infected plants senesced at 50 days after planting (DAP) as
opposed to 60 DAP in the uninoculated plants (data not shown). However, Pi did not
affect the growth period of other cultivars tested. In the exception of ‘Red Thumb’, plant
shoot growth and tuber quality were not different between M. incognita inoculated and
uninoculated plants. On the other hand, M. incognita inoculation resulted in difference in
TW and Rf, for all cultivars tested.
Tuber weight was compared among cultivars in the presence or absence of M.
incognita for both experiments (Table 3.1). Significant interaction among cultivars across
30
Figure3.1(A) Effect of Meloidogyne incognita population densities (Pi) on plant shoot
growth of red skinned potato cultivars 35 days after inoculation. Red Thumb plants
inoculated with 0, 5000 or 10,000 eggs/ plant, left to right, respectively.( B) effect of an
Pi 10,000 on plant shoot growth of cultivars, ‘Mountain Rose’, ‘Red Thumb’, ‘Desiree’
and ‘Pink Pearl’ (left to right).
A
B
31
Pi levels was observed in Trial 1 and Trial 2 (P ≤ 0.05). In Trial 1, TW was only suppressed by
M. incognita at the highest inoculation level (Pi = 2000 eggs/plant) on ‘Red Thumb’ (P < 0.05)
but not on other cultivars (Table 3.1). In Trial 2, TW of ‘Red Thumb’ was suppressed by Pi at
500 and 5000 eggs/plant (P ≤ 0.05). Rf also did differ among cultivars and Pi levels (Table 3.1).
Rf was consistently high (Rf >2) in ‘Desiree’, ‘Red Thumb’ and ‘Pink Pearl’ (Table 3.1).
‘Mountain Rose’ had the lowest Rf different from the others cultivars tested (P ≤ 0.05).
In all cultivars tested the total TW for Trial 2was much higher than TW for the Trial 1
(Table 3.1). Over all TW means comparison showed that Pink Pearl had the lowest TW, different
from the TW of the other cultivars (P≤0.05). TW differed among cultivars across nematode
population densities (Table 3.1). ‘Red Thumb’ also had a low TW but not different from
‘Desiree’ or ‘Mountain Rose’. TW was affected by Pi only in the case of ‘Red Thumb’.
Reduction in TW was observed in ‘Pink Pearl’ across nematode densities, but this was not
significant. No differences were detected in TW due to nematode population in ‘Desiree’ or
‘Mountain Rose’. TW of ‘Mountain Rose’ increased as nematode population increased (Table
3.1). Rf was the highest with ‘Pink Pearl’ and ‘Desiree’ (Rf > 18) and differed from that of ‘Red
Thumb’ and ‘Mountain Rose’ (P ≤ 0.01). The lowest Rf was detected in ‘Mountain Rose’ (Table
3.1). Only ‘Pink Pearl’ had a difference among Rf among Pis. Rf for Pi 500 was significantly
higher than Rf for Pi 5000 in ‘Red Thumb’. However, in most cases, Rf for Pi 5000 was the
highest. Although ‘Red Thumb’ had very low Rf compared to ‘Pink Pearl’ and ‘Desiree’, TW
was adversely affected by nematode population densities. The damage threshold density ( ≥10%
tuber weight reduction) was detected for ‘Red Thumb’ and ‘Pink Pearl’ at 10 and 100 nematode
eggs/250 cm3 soil, respectively (Fig. 3.2 A and B). However, damage threshold density was not
detected for ‘Desiree’ or ‘Mountain Rose’. ‘Mountain Rose’ on the
32
PiW
Desiree Mountain Rose Pink Pearl Red Thumb
TW Rfx
TW Rf TW Rf TW Rf
Trial 1 0 115 y
0 105 0 85 0 84 a 0
20 109 10 a 115 2 a 83 0.4 c 81 ab 27.0 a
200 128 6 ab 103 0 b 68 3.0 a 76 ab 2.2 b
2000 100 1 b 138 0 b 86 1.6 b 62 b 1.3 b
Average 113ze
6 j 115 e 0.6 k 81 f 1.6 k 76 f 10.0 i
Trial 2 0 223 0 201 b 0 177 0 219 a 0
500 209 25 199 b 2 161 12.0 b 223 a 5.1 a
5000 206 30 200 b 3 149 33.0 a 183 b 2.3 b
10,000 218 18 222 a 2 134 30.0 a 163 b 3.1 b
Average 214 e 24 i 205 e 2.3 j 155f 25.0 i 197 e 3.3 j
Table 3.1 Effect of different initial population densities (Pi) of M. incognita on tuber weight (TW) of re d
skinned potato and nematode reproductive ratio (Rf).
w Initial population (Pi) is number of M. incognita eggs/plant used for inoculation.
x Reproductive ratio Rf is calculated
by dividing final population (Pf) by Pi ( Rf= Pf/Pi). y Numbers within each column followed by same letter are not
significantly different according to Duncan’s MRT (P<0.05). z Numbers within average row (for comparing either TW
or Rf) followed by same letter are not significantly different according to Duncan’s MRT (P<0.05)
Cultivars
32
33
other hand, had the highest TW, even higher than TW in the uninoculated control, when
nematode inoculation was the highest (Pi= 10000 nematode/plant).
In most cases, ‘Red Thumb’ had a smaller root system and abnormally small
tubers compared to roots and tubers of nematode free plants (Fig. 3.3 A and B). A slight
reduction of TW was detected in ‘Pink Pearl’ due to M. incognita whereas plant growth
and tuber quality were not affected. Only tubers from ‘Red Thumb’ plants inoculated
with Pi 5000 or Pi 10,000 showed visible specks associated with nematodes (Fig. 3.3 C
and D). Very low numbers of eggs and J2s were detected in tubers of ‘Desiree’ and
‘Mountain Rose’ compared to ‘Pink Pearl’ and ‘Red Thumb’. Based on observation, J2s
from tubers of ‘Red Thumb’ and ‘Pink Pearl’ were active compared to starving or dead in
‘Desiree’ and ‘Mountain Rose’. Two males have been detected from two plants of
‘Mountain Rose’ but not from any other cultivar.
Discussion
Nematode population densities used in these experiments did not affect plant
growth in all cultivars tested in both experiments except in the case of ‘Red Thumb’.
Since reduction of plant shoot growth was only detected in ‘Red Thumb’ plants 35 after
inoculation with 5000 or 10000 eggs/ plant, and stunting, yellowing and severe wilting
were obvious on plants of this cultivar but not on other cultivars tested, our results
confirm the susceptibility and intolerance of ‘Red Thumb’ to M. incognita. The damage
threshold density of this cultivar is 10 nematode eggs/ 250 cm3 soil and that is higher than
for M. chitwoodi or M. hapla on potato (Santo et al., 1981; Brodie et al., 1993), but is
34
Figure 3.2 Damage thresholds for Meloidogyne incognita on red skinned potato cultivars.
The damage functions are derived by the polynomial regression of tuber weight reduction
percentage on initial population density (Pi) of M. incognita on ‘Red Thumb’ and ‘Pink
Pearl’, A and B respectively.
35
much lower than damage threshold of M. javanica on two white potato cultivars (Vovlas
et al., 2005). ‘Pink Pearl’ is probably moderately tolerant but highly susceptible cultivar.
Based upon TW and Rf (Canto-Saenz and Brodie, 1986), the damage threshold density of
‘Pink Pearl’ is 100 nematode eggs/250 cm3 soil. Damage threshold of ‘Pink Pearl’ was 10
fold of ‘Red Thumb’ indicating a much higher level of tolerance in ‘Pink Pearl’ to M.
incognita.
Our results showed no effect on TW due to nematode population densities in
‘Desiree’ and ‘Mountain Rose’, suggesting a high level of tolerance in both cultivars.
‘Desiree’ was not only tolerant but also highly susceptible. Desiree had an average Rf
greater than 1 in the two runs. Nematode population was suppressed and TW and TQ
were not affected by nematode densities in cultivar ‘Mountain Rose’. More over TW of
‘Mountain Rose’ consistently increased with increasing Pi suggesting not only high level
of tolerance but also probable resistance.
Although inoculation levels in the Trial 2 were much higher than in Trial 1,
overall TW of all cultivars tested in second run (winter time) was much higher than TW
in the first run (summer time). Obviously the environmental conditions such as cooler
temperatures and more frequent rainfall were important factors in improving potato yield
even though presence of relatively high population of plant-parasitic nematodes (Barker
and Olthof, 1976). Soil texture on the other hand may have increased potato yield in the
second run when a soil conditioner Big R was used as mulch and hilling agent, soil was
less impacted and better aerated compared to the soil used in first run (Midmore et al.,
1986).
36
Figure 3.3. Effect of M. incognita on plant root system and tuber quality of cultivar Red
Thumb. A and B, left, nematode free roots and tubers; Right, roots and tubers of plants
inoculated with 10,000 eggs+J2s of M. incognita. C and D, blemished tubers from plant
inoculated with 5000 and 10,000 eggs/plant, right to left respectively.
A
B
C D
37
The low numbers of eggs and unhealthy J2 in ‘Desiree’ and ‘Mountain Rose’
were probably because either the level of inoculations used in the two experiments was
not high enough, or the M. incognita have different responses and feeding favorability to
different parts of the plants (roots and tubers)(Brown et al., 2009).
It has been reported for infections of some crops that Rf was reduced with
increasing nematode inoculums densities (Di Vito et al., 1986; De Di Vito et al., 2004;
Vovlas et al., 2005). This may explain the decrease of Rf as Pi increased. Although the
total number of nematode was the highest when Pi was the highest, the highest Rf was at
the lower Pis, Rf declined at the highest Pi in all cultivars. However except in ‘Mountain
Rose’, Rf across Pi was consistently high indicating the good host status of the cultivars.
The high Rf (Rf >1) in ‘Mountain Rose’ was only detected in individual plants, 2 out of
12 plants, and only with lowest inoculums. This may indicate an acceptable level of
resistance to M. incognita.
The damage threshold M. incognita on ‘Desiree’ and ‘Mountain Rose’ to M.
incognita is surprisingly high. These cultivars demonstrate tolerance to M. incognita.
However, under conditions of our experiments, the damage threshold density (10% or
more of tuber weight reduction) to M. incognita was determined in cultivars ‘Pink Pearl’
and ‘Red Thumb’ to be 10 and 100 nematode eggs/250 cm3
soil respectively. TW and Rf
are efficient criteria to determine damage potential and host efficiency among red skinned
potato cultivars.
38
References
Barker, K.R. and Olthof, T.H.A. 1976. Relationships between nematode population
densities and crop responses. Annual Review of Phytopathology 14:327-353.
Brodie B. B., Evans, K., and Franco, J. 1993. Nematode parasites of potatoes. In: Evans,
K., Trudgill, D.L., and Webster J.M., eds. Plant Parasitic Nematodes in Temperate
Agriculture. Wallingford, UK: CAB International. pp. 87–132.
Brown, C. R., Mojtahedi, H., Zhang, L. H., and Riga, E. 2009. Independent resistant
reactions expressed in root and tuber of potato breeding lines with introgressed resistance
to Meloidogyne chitwoodi. Phytopathology 99:1085-1089.
Canto-Saenz, C. M., and Brodie, B. B. 1986. Host efficiency of potato to Meloidogyne
incognita and damage threshold densities on potato. Nematropica 16:109-161.
DiVito, M., Greco, N., and Carella, A. 1985. Population densities of Meloidogyne
incognita and yield of Capsicum annuum. Journal of nemtology 17:43-49
Di Vito, M., Greco N., and Carella A. 1986. Effect of Meloidogyne incognita and
importance of the inoculum on the yield of eggplant. Journal of Nematology 18: 487–90.
Di Vito, M., Vovlas, N., and Castillo, P. 2004. Host-parasite relationships of
Meloidogyne incognita on spinach. Plant Pathology 53:508–14.
Ferris, H., Carlson, H.H., and Westerdahl, B.B. 1994. Nematode population changes
under crop rotation sequences: Consequences for potato production. Agronomy Journal
68:340-348.
Hussey, R. S., and Barker, K. R. 1973. A comparison of methods of collecting inocula of
Meloidogyne spp. including a new technique. Plant Disease Reporter 57:1025-1028.
McSorley, R., Dickson, D.W., Candanedo-Lay, E.M., Hewlett, T.E. and Frederick, J.J.
1992. Damage functions for Meloidogyne arenaria on peanut. Journal of Nematology
24:193–198.
39
McSorley, R. and Parrado, J. L. 1981. Effect of sieve size on nematode extraction
efficiency. Nematropica 11:165-174.
Midmore,D. J., Roca, J. and Berrios, D. 1986. Potato (Solanum spp.) in the hot tropics
III. Influence of mulch on weed growth, crop development, and yield in contrasting
environments. Field Crops Research 15:109-124.
Noe, J.P. 1985. Analysis and interpretation of data from nematological experiments, p.
187–196. In: K.R. Barker, C.C. Carter, and J.N. Sasser (eds.). An advanced treatise on
Meloidogyne. Vol. II: Methodology. N.C. State Univ. Graphics, Raleigh.
Pinkerton, J.N. and Santo, G.S. 1986. Control of Meloidogyne chitwoodi in commercially
grown russet Burbank potatoes. Plant Disease 70:860-863.
Santo, G.S., O’Bannon, J. H., Nycepir, A. P., Ponti, R. P. 1981. Ecology and control of
root-knot nematodes on potato. Proceedings of the 20th Annual Washington Potato
Conference, 3–5 February, Moses Lake,Washington, USA. Moses Lake.
Vovlas, N., Mifsud D., Landa B. B., and Castillo, P. 2005. Pathogenicity of the root-knot
nematode Meloidogyne javanica on potato. Plant Pathology 54:657- 664.
40
CHAPTER 4. HOST EFFICIENCY AND HOST PARASITISM OF THREE
SPECIES OF MELOIDOGYNE ON RED SKINNED POTATO
Introduction
Meloidogyne incognita, M. javanica, and M. arenarea are the most important
species of RKN nematode in the tropics and subtropics causing severe damage on
economic crops including potato (Jatala, 1975; Taylor and Sasser, 1978). Knowing the
crop response to these nematodes is useful imformation to develop management program
against these RKN. Resistance genes to one species of Meloidogyne often can also confer
resistance to other species of RKN. For instance, bell pepper (Capsicum annuum) variety
resistant to M. incognita is resistant to M. javanica and M. hapla (Thies and Fery, 2000).
Sasser (1980) also reported that RKN resistance found in many crops offers resistance to
both M. incognita and M. javanica. Tomato (Solanum lycopersicum) can be resistant to
M. arenaria, M. incognita, and M. javanica by having only the Mi-1 resistant gene
(Dropkin, 1969). In most cases, potato with resistance to M. incognita has almost same
response to M. javanica (Greco et al., 2007). Similarly, it has been demonstrated that
Solanum species with resistance M. chitwoodi have resistance to M. fallax (Janssen et al.,
1995 and 1997; Van der Beek et al., 1998).
Several species of RKN commonly found in Hawaii are Meloidogyne javanica,
M. incognita and M. konaensis. M. javanica infects Carica papaya (papaya), Ananas
comosus (pineapple), Musa acuminatai (banana) and many vegetables (Blake, 1969;
Sipes et al., 2005; Vovlas et al., 2005). Meloidogyne konaensis is a problem in coffee
(coffea Arabica) but has a wide host range (Eisenback et al., 1994; Zhang and Schmittd,
41
1994). Meloidogyne incognita is found in roots of many vegetables as well as potato
(Jatala, 1975). To promote the cultivation of red skinned potato in Hawaii, more
information on behavior and response of this potato to M. incognita, M. javanica and M.
konaensis will be helpful for Hawaiian farmers especially if their fields are infested with
these RKN.
Two objectives in this thesis chapter were addressed. First, the susceptibility of
selected red skinned potato cultivars to M. incognita, M. javanica and M. konaensis.
Second, parasitism rate of M. incognita and M. javanica on red skinned potato cultivars
known to differ in susceptibility to M. incognita.
Materials and Methods
Susceptibility to Meloidogyne spp.
Meloidogyne incognita and M. javanica cultures were maintained in the
greenhouse on ‘Pixie’ tomato (Solanum lycopersicon cv.), and M. konaensis culture was
maintained on Guatemala tipica coffee (Coffea arabica). Egg inocula for all tests were
collected from 3-month-old tomato or 8-month-old coffee roots. Tomato roots were
gently washed with tap water, cut to 1-cm long piece and placed in a 500 ml flask with
0.5% NaOCl, whereas coffee roots were blended in a food processor with 0.5% NaOCl
for 10 seconds then poured in a 500 ml flask. The flasks were shaken for 4 minutes using
a wrist action shaker (Hussey and Barker, 1973). The liquid was passed through a 100-
µm pore sieve and collected on a 20-µm pore sieve after washing with tap water
(McSorley and Parrado, 1981). The eggs were counted and adjusted to 1000 eggs/ml.
42
A 44 factorial designed experiment (4 red skinned potato cultivars 4 RKN
inocula) conducted in pots was conducted at the University of Hawaii Magoon
Greenhouse facilities, Manoa, HI. The experiment was arranged in RCBD with four
replications. The 4 potato cultivars were ‘Desiree’, ‘Mountain Rose’, ‘Pink Pearl’, and
‘Red Thumb’ and inoculums were Meloidogyne incognita, M. javanica, M. konaensis,
or non-inoculated. Certified seed potatoes of the four cultivars were planted individually
in 6-liter paper-pulp pots. The pots were filled with 1:1:1 sterile sand, soil, and Big R (a
redwood soil conditioner, Kellogg Garden Products, Honolulu, HI). Plants were hilled
(with 2-3 cm of media) three times during the growing period. Pots were placed on raised
benches under the ambient conditions. Irrigation was applied as needed. Pre-plant
fertilization consisted of 16-16-16 All Purpose Gaviota (BEI Hawaii) fertilizer at 25g/pot,
followed by three additional 25 g/pot during the growing period. One week after planting,
the emerged plants were inoculated with a 10,000 eggs of M. incognita, M. javanica, or
M. konaensis in 10 ml nematode suspension/pot. Uninooculated control plants were
drenched with 10 ml fresh water/pot.
Plant height was recorded 35 days post inoculation (DPI). Potato tubers were
harvested after 50 days for Red Thumb and 70 days for the other three cultivars based on
the senesce time of that cultivar. Tuber weight and root weight were recorded for each
plant. Tubers were chopped into 1-cm3 pieces and blended in a food processor with 50 ml
0.5% NaOCl for 10 seconds, placed in 500 ml flasks, and shaken for 4 minutes in a wrist-
action shaker. Roots were carefully washed, placed in 500 ml flasks, and shaken with
0.5% NaOCl (Hussey and Barker, 1973). Eggs were collected by passing the liquid
through a 100-µm pore sieve and collecting eggs on a 20-µm pore sieve after washing
43
with tap water (McSorley and Parrado, 1981). Nematode numbers from tubers and roots
were counted under a dissecting microscope, and combined to give the final nematode
population density (Pf).
Tuber weight and nematode numbers were compared among cultivars and nematode
species. The Rf (Rf= Pf/Pi) was calculated for each cultivar-nematode combination, for
each nematode across cultivars, and for each cultivar across nematodes. Each comparison
was analyzed for differences. Tuber quality (TQ) was also compared among cultivars
and nematode densities using an infection index (Pinkerton et al., 1986) in a scale of 0-6,
0 = no infection, 1 = 1-3, 2 = 4-5, 3-6-9, 4 = 10+, 5 = 50+, and 6 = 100+ infection
sites/tuber. Data were log10 (x + 1) transformed to normalize the data before analysis
(Noe, 1985), and Waller-Duncan’s multiple range test was used for means separation
wherever appropriate.
Host parasitism
The host-parasite relationship was evaluated base on root penetration and
nematode development in two red skinned potato cultivars. Four plants of ‘Mountain
Rose’ and ‘Red Thumb’ were inoculated with a freshly hatched J2 of M. incognita or M.
javanica. The experiment was arranged in RCBD with four replications. Potato seed
tubers were planted in 2 liter pots containing 1:1:1 sand, soil, and Big R (Kellogg Garden
Products). Pots were maintained in a shade house and irrigated as needed. Ten-day-old
plants were inoculated with 1000 freshly hatched J2 of M. incognita or M. javanica. J2
were obtained from ‘Piexi’ tomato plants as previously described. Twelve DPI, plant
roots were gently washed free of soil and stained using methods described by Daykin and
Hussey (1985). Roots were cleared in a 100 ml beaker filled with 50 ml 5.5% NaOCl for
44
5 minutes. Roots were occasionally agitated while soaking. The roots were then rinsed
for 1 minute and soaked for 20 minutes in tap water to remove NaOCl residue. Roots
were transferred to beakers filled with 50 ml water and 1 ml acid-fuchsin-stain solution
(3.5g acid fuchsin/250 ml acetic acid and 750 ml distilled water). The root-solution was
boiled for 30 seconds, allowed to cool to room temperature, and rinsed with tap water.
Roots were distained in 30 ml of glycerin acidified with 5N HCl by heating to the boil.
Based on their amount, destained roots were divided to 3-4 portions, placed in inverted
Petri dishes, and nematodes were counted under the dissecting microscope. Nematode
numbers were recorded and differences between cultivars and Meloidogyne spp. were
compared. Developmental stages of the nematode inside the roots were also observed and
compared between nematode species across the cultivars (Abad et al., 2010). Data were
analyzed as a 22 factorial experiment and two means of nematode species were
compared for each cultivar.
Results
Susceptibility to Meloidogyne spp.
There were significant interactions between cultivars and Meloidogyne spp. (P <
0.01) in both TW and Rf. TW did not differ (P < 0.05) among cultivars (Fig. 4.1),
whereas Rf differed significantly among cultivars (P < 0.01) (Fig. 4.2). There were
significant (P < 0.01) effects of nematode species on, TW within cultivar, Rf within
cultivar and Rf among cultivars. TW of different potato cultivars to each RKN species
varied (Fig. 4.1). Tuber weight of ‘Desiree’ and ‘Red Thumb’ was reduced by all
nematode species. TW of ‘Mountain Rose’ was increased by M. incognita and M.
45
konaensis infection, but its TW was reduced by M. javanica. ‘Pink Pearl’ TW was
reduced by M. incognita (P ≤ 0.05), but was increased by M. javanica and M. konaensis.
Rf differed among cultivars and nematode species (Fig. 4.2). Pink Pearl had the
highest Rf among all four cultivars across nematode species (P < 0.05). Although Rf
among RKN species within cultivar ‘Desiree’ showed no significant differences, overall
Rf of M. incognita was higher (Rf > 4) than that of M. javanica (Rf > 3) while Rf of M.
konaensis was the lowest (Rf < 1) in this cultivar. For ‘Mountain Rose’, Rf of M.
javanica was higher (P ≤ 0.05) than those of M. incognita or M. komaensis. Rf did not
differ between M. incognita and M. komaensis in ‘Mountain Rose’. On ‘Pink Pearl’, Rf
of M. javanica did not differ significantly from that of M. konaensis, but both of them
Figure 4.1 Effect of three root-knot nematode species on tuber weight (TW) of four red
skinned potato cultivars. Light gray, white, black and gray bars present M. incocnita, M.
javanica, or M. konaensis, and no nematode, respectively. Within a cultivar, bars with
same letters are not different based on Waller-Duncan k-ratio (k=100) t-test.
b
a
d b
a
b
b
a a a
a
a a
a
c
a
0
50
100
150
200
250
300
Desiree Mountain Rose Pink Pearl Red Thumb
Tu
ber
wei
gh
t (T
W)
g
Mi Mj Mk Control
46
were much lower (P ≤ 0.05) than Rf of M. incognita (Fig. 4.2). On ‘Red Thumb’, Rf of
M. incognita was higher than Rf of M. javanica or M. konaensis (P ≤ 0.05), while Rf of
the last two was not different.
Host parasitism
Analysis of variance for this experiment showed very significant interaction
between cultivars and nematode species (P ≤ 0.01). A reversed trend on penetration rate
of M. incognita and M. javanica was observed between the two potato cultivars (Fig.
4.3). Penetration by M. incognita on cultivar ‘Mountain Rose’ was lower (P < 0.01) than
that by M. javanica (7% and 39% respectively), but penetration rate of M. incognita was
higher (P < 0.05) (38%) than M. javanica (13%) on ‘Red Thumb’. Meloidogyne javanica
on the other hand had higher penetration rate (P < 0.01) on ‘Red Thumb’ (13%) than M.
incognita on ‘Mountain Rose’ (7%).
Nematode development was affected by nematode species and cultivar. On
‘Mountain Rose’, 12 DPI, low numbers of M. incognita had penetrated and these
nematodes were developing slowly. Most of these nematodes were still vermiform J2. No
multiple infections were detected (Fig4.4, A and C). Unlike M. incognita, M. javanica on
the ‘Mountain Rose’ had a high number of J2 inside the roots. These nematodes had a
faster development already becoming swollen J2s. Root swellings were larger and
multiple infections were frequently detected (Fig. 4.4, B and D). Meloidogyne incognita
had higher penetration and faster development on ‘Red Thumb’ compared to M. javanica.
M. incognita on ‘Red Thumb’ was mostly detected at fourth stage juvenile (J4) (Fig. 4.5).
No difference in root swelling on ‘Red Thumb’ was evident between the different
nematode species (Fig. 4.6, A and B). Multiple infections in ‘Red Thumb’ were more
47
Figure 4.2 Reproduction factor (Rf) among four red skinned potato cultivars inoculated
with 10,000 eggs of M. incocnita (Mi), M. javanica (Mj) or M. konaensis (Mk). Within a
cultivar, bars with same letters are not different (P≤0.05) based on Waller-Duncan
grouping.
Figure 4.3 J2 penetration at 12 DPI of M. incognita and M. javanica inoculated to
Mountain Rose and Red Thumb red skinned potato. Symbols (**) indicate difference (P
≤ 0.01) between means within cultivar. Letters (a and b) indicate difference (P ≤ 0.01)
between means on different cultivars.
a
b
a
a a
a
b
b a b b
b 0
2
4
6
8
10
12
14
16
18
20
Desiree Mountain Rose Pink Pearl Red Thumb
Rep
rod
uct
ion
fact
or
(Rf)
Mi Mj Mk
b
** **
a
0
10
20
30
40
50
Mountain Rose Red Thumb
Pen
etra
ted
nem
ato
de
%
M. incognita
M. javanica
48
frequently detected in plants inoculated with M. incognita than with M. javanica (Fig.
4.6, C and D). Unlike M. javanica, M. incognita was not always detectable inside the
developing galls especially in ‘Mountain Rose’.
Figure 4.4 Development and gall size comparison between Meloidogyne incognita and
M. javanica on red skinned potato cultivar Mountain Rose at 12 DPI. M. incognita
showed slow development and abnormal gall formation (A and C, respectively). M.
javanica developed faster with relatively bigger gall formation (B and D respectively).
49
Figure 4.5 Developmental stages of Meloidogyne incognita. (A) Developmental stages,
from eggs to adult nematodes (from Abad et al., 2010). (B) Early developing J4 of M.
incognita from Red Thumb roots 12 DPI.
Figure 4.6 Development and gall size comparison between Meloidogyne incognita and
M. javanica on red skinned potato cultivar Red Thumb at 12 DPI. Meloidogyne incognita
showed normal gall formation and frequent multiple infection (A and C, respectively). M.
javanica had similar gall formation (B), but less frequent multiple infections (D).
50
Discussion
‘Red Thumb’ is susceptible and intolerant to M. incognita (Chapter 2 and 3).
Although Red Thumb is resistant to M. javanica and M. konaensis, its TW was decreased
by these nematodes suggesting its intolerance to both of these species. ‘Desiree’ was not
always tolerant to M. incognita (Chapter 2 and 3), but was moderately tolerant to both M.
javanica and M. konaensis. ‘Mountain Rose’ was tolerant and moderately resistant to M.
incognita infections confirming previous results. ‘Mountain Rose’ was also tolerant and
resistant to M. konaensis. However ‘Mountain Rose’ was susceptible and intolerant to M.
javanica. Interestingly ‘Pink Pearl’ is an efficient susceptible with different levels of
tolerance to all three nematode species. ‘Pink Pearl’ was very susceptible – intolerant to
M. incognita, but susceptible/tolerant to both M. javanica and M. konaensis. ‘Red
Thumb’ was the most susceptible/intolerant to M. incognita, and the most resistant with
moderate tolerance to both M. javanica and M. konaensis, among all cultivars tested.
‘Mountain Rose’ behaved adversely to M. javanica compared to M. incognita.
Meloidogyne incognita was the most aggressive nematode on all cultivars tested
based upon Rf and TW reduction. Cultivar with resistance to M. incognita will often be
resistant or moderately resistant to M. javanica (Greco et al., 2007; Sasser, 1980; Thies
and Fery, 2000). However, this case did not fit our findings in which M. incognita was
suppressed by ‘Mountain Rose’ with no effect on TW but M. javanica efficiently
reproduced and significantly decreased TW on same cultivar. This was also demonstrated
by the penetration and development between the two nematode species were compared
between the two cultivars.
51
Penetration and development confirmed the differences between ‘Mountain Rose’
and ‘Red Thumb’. In case of ‘Mountain Rose’, M. incognitai J2s were not always
detectable inside the developing galls. ‘Mountain Rose’ may have a hypersensitive
reaction (HR) against M. incognitai at the infected root cells (Haynes and Jones, 1976),
or the J2s of M. incognitai may have exited from the roots as a results of failing to
establish a feeding site (Anwar and McKenry, 2002). Meloidogyne incognita resistant
Medicago truncatula plants showed that HR or necrosis are not always evidences of
resistance to nematodes. The infective J2 in some cases may die or develop as males
without distinguishable HR reaction (Dhandaydham et al., 2008; Trudgill, 1967). This
may explain former observations (Chapter 3) in which we found only males in tubers
from two ‘Mountain Rose’ plans infected with M. incognita, while no male was detected
from any other nematode species on any of other three cultivars.
The slow development of M. incognita on ‘Mountain Rose’ may suggest a
genetically specific interaction or possible involvement of resistance genes such as N
gene or R genes. Cowpea plants with R-gene Rk prevented developing J2 from reaching
maturity (Das et al., 2008). Thus mechanisms of resistance especially in case of plant
interactions with nematode seem to be diverse and complex.
Interestingly we found a unique relationship or genetically specific interaction
between ‘Mountain Rose’ and M. incognita as well as between ‘Red Thumb’ and M.
javanica. ‘Red Thumb’ may be cultivated in a field infested with M. javanica in order to
suppress this nematode and mitigate yield loss. Similar approach can be used in case of
‘Mountain Rose’ and M. incognita. It also might be useful to rotate ‘Red Thumb’ with
‘Mountain Rose’ where the field infested with mix population of both M. incognita and
52
M. javanica. Further understanding to such specific resistance might be useful and
practical in breeding potato resistant to multiple species of Meloidogyne especially those
are economically important in tropics and subtropics.
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