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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