evaluation of root-knot nematodes (meloidogyne...

EVALUATION OF ROOT-KNOT NEMATODES (Meloidogyne spp.) ON PEACH (Prunus persica) IN FLORIDA

By

SAI QIU

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

To my parents, my husband, and my dear daughter, Flora, for their love, consideration, and support.

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ACKNOWLEDGMENTS

I would like to give my deepest appreciation to Dr. Donald W. Dickson, who is the

best advisor and mentor in my mind. He was consistently helping me with my project

and supporting me during the past two years. In order to prepare for my materials, he

spent days helping me crack the peach seeds and rooting peach cuttings. In order to

help me maintain a suitable environment for my plants, he was installing the irrigation

system when the temperature was more than 40 °C. As I had field experiment in South

Florida, he spent his precious time driving the truck and helping me to collect samples

from early morning to evening. He was always the person standing by me and giving

me help. During the past two years, I learned a lot from him. As a Nematologist, he

expanded my view of Nematology and agriculture. As a group leader, he taught me how

to treat group members with love and cooperate efficiently. As an advisor, he showed

me that students’ work was always his priority. As my instructor of the course ‘Plant

nematology’, he trained me to have the basic skills of doing research.

I would like to thank my committee members, Drs. Jose Chaparro, Janete Brito,

and Larry Duncan, who were excellent scientists and worked together as a family to

support my project. Without their passionate participation and input, the validation

survey could not have been successfully conducted. Dr. Chaparro did his best to make

plant materials available for my experiment and the door to his office was always open

for me whenever I had questions. He and Dr. Brito were supportive of my project. They

often went with me to visit peach farms and helped me to choose the ideal sampling

sites. Dr. Brito, like my mother in this country, always treated me as her daughter in my

life and gave me continuous guidance in my project. I would like to acknowledge Dr.

Duncan for his valuable suggestions and comments on my project.

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I would like to express my gratitude to Dr. Tom Beckman from USDA and Dr.

Mercy Olmstead, the University of Florida. Without them providing plant materials, my

experiment could not have been conducted.

I would also like to thank my dear lab mates, Silvia Vau and Weimin Yuan, who

did a lot to make the lab like a family. I would like to thank my dear friends, Dr. Maria

Mendes and Ms. Mary Ann Maquilan, for their help and friendship.

Finally, I would like to thank my parents, my mother-in-law, my father-in-law for

their help and encouragement. In addition, I would appreciate my husband, and my

adorable daughter for their love and support.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 8

LIST OF FIGURES .......................................................................................................... 9

ABSTRACT ................................................................................................................... 11

CHAPTER

1 INTRODUCTION .................................................................................................... 12

History and Geographical Distribution of Root-Knot Nematodes ............................ 12

Life Cycle and Mode of Reproduction ..................................................................... 13

Diseases and Damage............................................................................................ 14

Root-Knot Disease on Peach and Management ..................................................... 15

Objectives ............................................................................................................... 17

2 MATERIALS AND METHODS ................................................................................ 18

Greenhouse Experiment ......................................................................................... 18

Nematode Source ............................................................................................ 18

Identification and Characterization of Meloidogyne spp. .................................. 19

Preparation of Peach Seedlings ....................................................................... 19

Host Differential Test ........................................................................................ 20

Evaluation of Root-knot Nematode Infection and Reproduction on Peach Rootstocks .................................................................................................... 21

Data and Statistical Analysis ............................................................................ 23

Field Experiment ..................................................................................................... 23

Site Locations ................................................................................................... 23

Depth Sampling ................................................................................................ 23

Soil Analysis ..................................................................................................... 24

Data and Statistical Analysis ............................................................................ 25

3 RESULTS AND DISCUSSION ............................................................................... 27

Greenhouse Experiment ......................................................................................... 27

Host Differential Test ........................................................................................ 27

Experiment 1 .................................................................................................... 27

Experiment 2 .................................................................................................... 28

Experiment 3 .................................................................................................... 29

Experiment 4 .................................................................................................... 31

Field Experiment ..................................................................................................... 32

Site Description and Selection .......................................................................... 32

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Population Densities of Root-Knot, Ring, and Lesion Nematodes at Pampered Peach and Capron Trail Farms. ................................................... 33

APPENDIX: ISOENZYME ELETROPHORENSIS FOR THE IDENTIFICATION OF Meoidogyne spp. .................................................................................................... 63

LIST OF REFERENCES ............................................................................................... 64

BIOGRAPHICAL SKETCH ............................................................................................ 72

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LIST OF TABLES

Table page 2-1 The origin of root-knot nematode isolates used in this study. ............................. 26

3-1 Host differential test of five isolates of Meloidogyne floridensis (Mf) and two isolates of M. arenaria (Ma). ............................................................................... 37

3-2 The reproduction of four isolates of Meloidogyne floridensis (Mf) and two isolates of M. arenaria (Ma) on three peach cultivars: Flordaguard, Okinawa, and Lovell. .......................................................................................................... 38

3-3 The reproduction of two isolates of Meloidogyne javanica (Mj) and two isolates of M. incognita (Mi) on three peach cultivars: Flordaguard, Okinawa, and Lovell. .......................................................................................................... 39

3-4 The effect of three inoculum levels of Meloidogyne floridensis isolate (Mf5) collected from peach cultivar Flordaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., 2014 on three peach cultivars ................................. 40

3-5 Results of the main effects ANOVA for the of three inocula levels of Meloidogyne floridensis isolate Mf5 on three peach cultivars: Flordaglo, Okinawa, and Flordaguard. ................................................................................ 41

3-6 The reproduction of three isolates of Meloidogyne floridensis (Mf) on three peach cultivars: Flordaguard, Okinawa, and Lovell. ........................................... 42

3-7 The reproduction of Meloidogyne arenaria isolates Ma1 on three peach cultivars: Lovell, Okinawa, and Flordaguard and Ma2 on peach cultivar Flordaguard. ....................................................................................................... 43

3-8 Number of eggs per egg mass and range produced by Meloidogyne spp. on peach rootstock cvs. Flordaguard and Lovell. .................................................... 44

3-9 The reproduction of one isolates of Meloidogyne floridensis and two isolates of Meloidogyne arenaria on peach cultivar Flordaguard. .................................... 45

3-10 Soil characteristics of the soil depths sampled at Pampered Peach and Capron Trial Farms. ............................................................................................ 46

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LIST OF FIGURES

Figure page 3-1 Gall index responses of inoculum levels to peach rootstock cultivars. ............... 47

3-2 Egg mass index responses of inoculum levels to peach rootstock cultivars. ...... 48

3-3 Number of root-knot nematode eggs per gram fresh root weight responses of inoculum levels to peach rootstock cultivars. ...................................................... 49

3-4 Reproductive factor responses of inoculum levels to peach rootstock cultivars. ........................................................................................................................... 50

3-5 Galling on peach rootstock cv. Lovell roots caused by Meloidogyne floridensis isolate Mf2, that was originally collected from tomato grown at the Syngenta Research Farm, Indian River Co., FL.. ............................................... 51

3-6 Galling on peach rootstock cv. Lovell roots caused by Meloidogyne floridensis isolate Mf4 (topotype), that was originally collected from peach rootstock cv. Nemaguard at the Peach Research Orchard ................................ 52

3-7 Galling on peach rootstock cv. Flordaguard roots caused by Meloidogyne arenaria isolate Ma2, that was originally collected from peach at the Rafool Farm, Polk Co., FL. ............................................................................................ 53

3-8 Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne arenaria females extracted from roots of peach collected from Pampered Peach Farm, Polk Co., FL. ................................................................ 54

3-9 Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne floridensis females extracted from roots of peach collected from Capron Trail Farm, St. Lucie Co., FL. ................................................................. 54

3-10 Seasonal changes in the mean number of Meloidogyne arenaria second-stage juveniles (J2) extracted from 200 cm3 of soil collected from four soil depths at the Pampered Peach Farm, Polk Co. FL.. .......................................... 55

3-11 Seasonal changes in the mean number of Mesocriconema ornatum extracted from 200 cm3 of soil collected from four soil depths at the Pampered Peach Farm, Polk Co. FL. Each data point was the mean of 12 samples. .................... 56

3-12 Seasonal changes in the mean number of Pratylenchus hippeastri extracted from 200 cm3 of soil collected from four soil depths at the Pampered Peach Farm, Polk Co. FL.. ............................................................................................ 57

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3-13 Seasonal changes in the mean number of Meloidogyne floridensis second-stage juveniles (J2) extracted from 200 cm3 of soil collected from two soil depths at the Capron Trial Farm, St. Lucie Co. FL. ............................................ 58

3-14 Seasonal changes in the mean number of ring nematodes Mesocriconema xenoplax extracted from 200 cm3 of soil collected from two soil depths at the Capron Trial Farm, St. Lucie Co. FL. .................................................................. 59

3-15 Seasonal changes in the mean number of Pratylenchus brachyurus extracted from 200 cm3 of soil collected from two soil depths at the Capron Trial Farm, St. Lucie Co. FL. ................................................................................................. 60

3-16 Seasonal changes in the mean number of root-knot nematode eggs and root-knot nematode second-stage juveniles (RKN J2) extracted per gram of peach roots collected from Pampered Peach Farm, Polk Co. FL.. ..................... 61

3-17 Seasonal changes in the mean number of root-knot nematode eggs and root-knot nematode second-stage juveniles (RKN J2) extracted per gram of peach roots collected from Capron Trial Farm, St. Lucie Co. FL. ....................... 62

A-1 Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne arenaria females extracted from tobacco roots. Ma1 was collected from peach at Pampered Peach Farm, Polk Co., FL. .......................... 63

A-2 Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne arenaria females extracted from pepper roots. Ma2 was collected from peach at Rafool Farm, Polk Co., FL.. .......................................... 63

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

EVALUATION OF ROOT-KNOT NEMATODES (Meloidogyne spp.) ON

PEACH (Prunus persica) IN FLORIDA

By

Sai Qiu

May 2017

Chair: Donald W. Dickson Major: Entomology and Nematology

Meloidogyne floridensis, the peach root-knot nematode, is only reported to occur

in Florida and is considered as a potential threat to the newly developing peach industry.

On numerous occasions, this nematode and M. arenaria were isolated from commercial

peach orchards. The objectives of this project were to determine whether M. floridensis

and M. arenaria infect the root-knot nematode resistant peach rootstock cv. Flordaguard

and to follow the seasonal population changes of these two nematode species in two

commercial peach orchards. Of four isolates of M. floridensis, only one that was recently

extracted from Flordaguard consistently infected this rootstock in greenhouse

experiments. Two isolates of M. arenaria recently collected from peach were shown to

infected Flordaguard. This is the first report of M. arenaria infecting peach in Florida.

Based on differential host test, both M. arenaria species were designated as race 3.

Peach rootstock cv. Okinawa was found to be a nonhost of both M. floridensis and M.

arenaria. During a 17-month sampling period at two peach orchards, the number of

eggs and second-stage juveniles of M. arenaria and M. floridensis reached the highest

number in August 2015 and March 2016. The population density, however, declined at

both sites during the last 6 months of the sampling period.

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CHAPTER 1 INTRODUCTION

History and Geographical Distribution of Root-Knot Nematodes

Root-knot nematode was first reported by Berkeley in 1855, when he described

root-knot nematodes on cucumber (Cucumis sativus) in England (Berkeley, 1855). The

second reported root-knot nematode, Meloidogyne exigua Göldi, 1887, was found

infecting coffee roots in Brazil. After that, an increasing number of root-knot nematode

species were discovered. By 2009, more than 94 root-knot nematode species have

been described worldwide (Layne and Bassi, 2008).

Root-knot nematode species within the genus have different geographical

preferences based on their sensitivity to temperature and other ecological factors

(Walters and Barker, 1994). There are four major root-knot nematode species that are

distributed worldwide affecting a wide array of important agriculture crops. Three of

these species, M. incognita Kofoid and White, 1919, M. javanica Treub, 1885, and M.

arenaria Neal, 1898 are commonly found in warmer regions, whereas M. hapla

Chitwood, 1949 is adapted to and survives overwinter in cooler regions, such as the

northern United States, southern Canada, and Slovakia in Europe (Walters and Barker,

1994; Lišková and Sasanelli, 2007). However, some newly described root-knot

nematode species, e.g., M. baetica Castillo, Vovlas, Subbotin, and Troccoli, 2003 and M.

floridensis Handoo, Nyczepir, Esmenjaud, van der Beek, Castagnone-Sereno, Carta,

Skantar, and Higgins, 2004 may be less widespread or are found in restricted regions

(Castillo et al., 2003; Handoo et al., 2005).

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Life Cycle and Mode of Reproduction

Root-knot nematodes are sessile endoparasites and require feeding on plant

roots to complete their life cycle. The cycle of root-knot nematodes includes one egg

stage, four juvenile stages, and an adult stage. The eggs, which are laid by the mature

female, are retained inside a gelatinous matrix commonly referred to as an egg mass.

The first-stage juvenile (J1) develops within the egg and the first molt occurs in the egg

giving rise to a second-stage juvenile (J2). Once the J2 hatches from the egg, this

infective stage juvenile seeks out plant roots and penetrates at or near root tips. The J2

migrates within the root cortex of the elongation region, eventually ending up with its

head end within the vascular system. The J2 establishes a specialized feeding site that

enables it to continue to develop through the third, and forth stages, and after the final

molt the female or male is formed. The female lives as a sedentary parasite. The

feeding site becomes a multinucleated giant cell, that is formed by nuclear divisions

without cell division (Jones and Payne, 1978). The cortical cells surrounding the female

also increase in number that results in the formation of a gall on knot on the root.

The mode of reproduction of root-knot nematodes is by parthenogenesis or

amphimixis, however in one case one population of M. hapla was reported to reproduce

by hermaphroditism (Triantaphyllou, 1993). Parthenogenesis does not involve males or

male gametes in fertilization, but amphimixis requires both male and female mating to

complete the fertilization process. According to Castagnone-Sereno (2006), the

nematode’s ability to reproduce parthenogenetically and their rapid adaptive responses

to varying environmental conditions have contributed to their successful establishment

as plant parasites with wide host ranges as well as with a wide geographical distribution.

Meloidogyne incognita, M. arenaria, and M. javanica, all of which occur in Florida, are

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known to reproduce by obligatory mitotic parthenogenesis (Baum, 1994; Block, 1997;

Castagnone-Sereno et al., 1994). These mitotic parthenogenetic species are

considered to be closer to each other evolutionarily than to amphigenetic species like M.

hapla (De Ley et al., 2002). However, the mode of reproduction of M. floridensis is not

unknown (Handoo et al., 2004).

Diseases and Damage

The disease caused by root-knot nematodes is commonly referred to as root-

knot (Zuckerman et al., 1993). Infected plants show belowground and aboveground

symptoms. The aboveground symptoms include incipient wilting with a slow recovery

with improved soil moisture conditions, chlorosis, stunting, and lacking vigor. The

belowground symptom of root-knot is galls on roots induced by the females (Dropkin,

1972; Sijmons et al., 1994). The size and the number of galls on roots vary depending

on the host plant and the root-knot nematode species (Bird, 1974). For example, in

susceptible tobacco cultivars, M. javanica induces larger galls than M. incognita and M.

arenaria (Arens et al., 1981).

The presence of root galls on plant roots is more reliable than aboveground

symptoms for the diagnosis of root-knot. Aboveground symptoms are similar to those

caused by other root diseases and by abiotic factors, such as drought and nutrient

deficiency. Aboveground symptoms alone cannot confirm root-knot, however, the galls

and egg masses on roots are helpful for diagnosing the disease.

Different species of root-knot nematodes are known to have varying host ranges

that results in them causing serious economic losses in tropical and sub-tropical regions.

They not only infect agricultural crops (Koenning et al., 1999), but they also increase on

numerous herbaceous plants (Ehwaeti et al., 1999), and weeds (Rich et al., 2009).

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Root-knot nematodes can damage plant roots in many types of soil (Koenning et al.,

1996; Windham and Barker, 1986); however, numerous studies show they prefer sandy

soils as compared with those containing a higher percentage of clay (Sleeth and

Reynolds, 1955; Barker and Weeks, 1991; Divito et al., 1985).

Although root-knot nematodes cause serious damage to numerous plant species,

it remains difficult to give accurate estimates of yield suppression. The mechanical

damage of root-knot nematodes to plants starts immediately once roots are penetrated;

after which, plant roots undergo continuous morphological changes as the J2 migrates

within the cortex tissue. Since they establish a feeding site, a gall is induced and the

females lays eggs. The eggs hatch resulting in repeated infection from newly hatched

J2 (Caillaud et al., 2008). There are considerable economic losses that results from the

direct decrease of crop productivity as well as an indirect loss from the wasted irrigation

water and nutrients that are required for growing the crop (Wesemael et al., 2011; Mai,

1985).

Root-Knot Disease on Peach and Management

Root-knot nematodes are considered an important limiting factor in peach

(Prunus persica [L.] Batsch) production. In the Mediterranean region of Europe, the

most common root-knot nematode species reported infecting peach are M. arenaria, M.

incognita, and M. javanica (Fernandez et al., 1994). In France, Portugal, Spain, and The

Netherlands, the most common species on peach is M. hispanica Eisenback, Bernard,

Starr, Lee, and Tomaszewski, 2003. Meloidogyne exigua has been reported on peach

in Greece and Italy (Wesemael et al., 2011). In North Africa, M. morocciensis Rammah

and Hirschmann, 1990 was found infecting peach rootstock. In the southern US, the

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most common root-knot nematode species infecting peach are reported as M. incognita

and M. javanica (Nyczepir, 1991).

In Florida, peach is being promoted as an alternative crop for the declining citrus

industry, and numerous low chill peach cultivars have been made available (Olmstead

et al., 2007; Brito et al., 2015). Also, peach rootstocks with root-knot nematode

resistance have been developed and are considered important for the sustainability of

the industry. The peach rootstock cvs. Nemaguard, and Nemared, which were released

by USDA, and Okinawa introduced from Japan were reported as resistant to M.

incognita, M. javanica, and M. arenaria (Okie et al., 1985; de Paula et al., 2011). Among

these three root-knot nematode resistant rootstocks, Nemaguard was planted

extensively in Florida during the industry’s introduction (Sherman and Lyrene, 1983).

However, both Nemaguard and Okinawa were soon found to be damaged by root-knot

nematodes. Originally, at least in one instance, the species was erroneously identified

as M. incognita race 3 (Sharpe et al., 1969). Later, Nemared was also found to be

infected by the same species (Sherman et al., 1991). Further investigations led to it

being identified as a new species and named the peach root-knot nematode, M.

floridensis (Handoo et al., 2004). Because of the discovery of M. floridensis infecting

Okinawa, Nemaguard, and Nemared rootstocks, these three rootstocks were no longer

suggested as a good choice for commercial production in Florida. A new peach

rootstock cv. Flordaguard, which was believed to be resistant to M. floridensis, M.

javanica, and M. incognita was released for combating the peach root-knot nematode

problem (Sherman et al., 1991).

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Shortly after Flordaguard was released as the suggested root-knot nematode

resistant rootstock, reports begin to surface that it was being damaged by root-knot

nematodes. Field sampling of commercial orchards revealed that peach was infected by

both M. floridensis and M. arenaria.

Objectives

The goal of this project was to evaluate the susceptibility of Flordaguard

rootstock to M. floridensis and M. arenaria isolates that were collected in Florida, and to

determine the seasonal fluctuation of field populations of root-knot nematodes on peach

in two different soil types and locations in Florida.

The objectives were to: 1) determine the degree of infection of the peach

rootstock cvs. Flordaglo, Okinawa, and Flordaguard to different inocula levels of the M.

floridensis collected from peach in 2014; 2) determine infection of M. floridensis and M.

arenaria isolates, which were collected from field plantings, on peach rootstock cvs.

Lovell, Okinawa, and Flordaguard; 3) determine the seasonal population fluctuations of

root-knot nematodes in two different peach orchards at four soil depths in central Florida

and at two soil depths in south Florida during a period of 17-months; and 4) determine

the soil elements and percentages sand, silt, and clay in the two peach orchards.

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CHAPTER 2 MATERIALS AND METHODS

Greenhouse Experiment

Nematode Source

Five isolates of Meloidogyne floridensis (Mf), two isolates each of M. arenaria

(Ma), M. javanica (Mj), and M. incognita (Mi) were used in this study. Four isolates of M.

floridensis were obtained from a root-knot nematode collection maintained by the

Division of Plant Industry, Florida Department of Agriculture and Consumer Services,

Gainesville, FL (Brito et al., 2007), and one isolate was collected from the University of

Florida Fruit Teaching Orchard, Alachua Co., FL. M. arenaria and M. javanica isolates

were collected from field plantings of peach.

The designation, host plant, and origin of each Meloidogyne spp. used in this

project are as follows: Mf1 (N05-227-17B), tomato, Seminole Co.; Mf2 (N03-1582-2B),

tomato, Indian River Co.; Mf3 (N04-627-5B), cucumber, Hendry Co.; Mf4 (N03-1894,

topotype), peach rootstock cv. Nemaguard, University of Florida Fruit Teaching Orchard,

Gainesville; Mf5 (PS-4), peach rootstock cv. Flordaguard, University of Florida Fruit

Teaching Orchard, Gainesville; and Ma1 (PS-1567) and Ma2 (PS-19), peach, Polk Co.,

FL. Mj1 (PS-69), and Mj2 (IFC-1) were collected from peach, Pasco Co., FL., and

Alachua Co., FL., respectively. Mi1, and Mi2 were collected from cotton, Decatur Co.,

GA., and weed Alachua Co., FL., respectively (Table 2-1).

All isolates were reared separately on tomato cv. Agriset 334 grown in steam

pasteurized soil and maintained in a greenhouse. All plants were watered daily and

fertilized weekly with 4.5 g/l Miracle Grow (Marysville, OH).

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Identification and Characterization of Meloidogyne spp.

The identities of M. floridensis and M. arenaria isolates were confirmed based on

their isozyme phenotype profiles (Dickson et al., 1971; Esbenshade and Triantaphyllou,

1985), and their race designation was determined by differential host tests (Taylor and

Sasser, 1978; Fargette, 1987).

Preparation of Peach Seedlings

Peach rootstocks came from three sources. Source one: Peach rootstock

Flordaglo, Okinawa, and Flordaguard semi-hardwood cuttings were harvested in June

and July of 2014 from the University of Florida Fruit Teaching Orchard, Gainesville, FL.

Cuttings with two leaves were treated with the plant hormone Hormodin 2 (Mainland, PA)

and set in a 32 cell plastic growing tray containing a steam-pasteurized potting mixture

(three parts Fafard 2B mix: one part perlite [v/v]). Trays were then placed in a

greenhouse under a mist propagation unit. Once rooted, the cuttings were transplanted

October 2014 into clay pots (16-cm diam.) filled with peat, and held overwinter in the

greenhouse. April 2015, the seedlings were transplanted into clay pots (25-cm diam.)

with three parts sand: one part perlite (v/v) mixture that had been steam-pasteurized.

Source two: Seeds of the peach rootstock cv. Lovell were obtained from the

USDA/ARS Southeastern Fruit and Nut Tree Research Laboratory, Byron, GA, and the

seeds of Okinawa and Flordaguard were obtained from the Horticultural Sciences

Department, University of Florida, Alachua Co., FL. All seeds were harvested in 2015.

Peach seeds were soaked in water for 3 days and rinsed with running water every 24

hours before being treated in captan with 2 g/L (Oriskyany, NY) at room temperature

overnight. All seeds were rinsed with running water to remove the fungicide, and

stratified in perlite in zip-lock bags and stored in a chamber (7 °C) for 60 to 90 days

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(Olmstead et al., 2007). After achieving dormancy breaking, seeds were planted into 32-

cell plastic growing trays (5 cm × 5 cm × 7.5 cm square cells) containing steam-

pasteurized Fafard 2B mix (Sun Gro Horticulture, Agawam, MA) and held in a

greenhouse to induce germination. When the seedlings were ca. 20 cm tall, they were

transplanted into clay pots (25-cm diam.) containing two parts sand: one-part coarse

vermiculite: 1 part perlite (v/v) steam-pasteurized mixture. Seedlings were maintained in

a greenhouse for 1 month before inoculation.

Source three: One-year old Flordaguard seedlings were provided by Island

Grove AG Products: nursery division, Island Grove, FL and maintained in 3.5-L plastic

pot in the greenhouse with one-part sand: four parts peat (v/v) mixture. All seedlings

were grown from cuttings harvested from Flordaguard trees that had been verified as

true to type by a DNA marker test (Maquilan, 2014).

All peach seedlings were watered daily and fertilized weekly with 4.5 g/l Miracle

Grow.

Host Differential Test

All inocula were obtained from infected tomato roots. Eggs were extracted with a

solution of 0.25% NaOCl (Boneti and Ferraz, 1981). Before inoculation, the inocula

were diluted to a concentration of 1,000 eggs and (or) J2/ml suspension. The egg

suspension was applied to four equally spaced holes punched 3 cm deep around the

plant stem base.

All isolates of M. floridensis and M. arenaria were subjected to host differential

test conducted in the greenhouse using a protocol as previously described (Taylor and

Sasser, 1978). Plant species and cultivars used in the test were: tobacco (Nicotinana

tabacum cv. NC95), cotton (Gossypium hirsutum cv. Deltapine 16), pepper (Capsicum

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annuum cv. California Wonder), watermelon (Citrullus lanatus cv. Charleston Grey),

peanut (Arachis hypogaea cv. FL 07), and tomato (Solanum esculentum cv. Agriset

334). Each plant was inoculated with 5,000 eggs and (or) J2 of each of the isolates to

be tested. All plants were maintained in a greenhouse and arranged on benches in a

completely randomized design.

Evaluation of Root-knot Nematode Infection and Reproduction on Peach Rootstocks

In experiment 1, three peach rootstocks Flordaglo, Okinawa, and Flordaguard

were inoculated with 5,000 eggs and (or) J2/plant of each of four M. floridensis (Mf1,

Mf2, Mf3, and Mf4), two M. arenaria (Ma1 and Ma2), two M. javanica (Mj1 and Mj2), and

two M. incognita (Mi1 and Mi2) isolates. Each treatment combination had four

replications and each plant was placed in a 1-L plastic pot.

In experiment 2, the effects of three inoculum levels (2,000, 5,000, and 10,000

eggs and [or] J2/plant) of M. floridensis (Mf5) were tested on three peach rootstocks,

Flordaglo, Okinawa, and Flordaguard. The treatments were replicated four times and

plants were placed in clay pots (25-cm diam.).

In experiment 3, the infection of Lovell, Okinawa, and Flordaguard by three M.

floridensis isolates (Mf1, Mf2, and Mf4) and two M. arenaria isolates (Ma1 and Ma2)

were tested. In April 2016, peach rootstocks were inoculated with 10,000 eggs and (or)

J2/plant of each isolate and replicated seven times. Each plant was placed in a clay pot

(16-cm diam.).

In experiment 4, one M. floridensis isolate (Mf5) and two M. arenaria isolates

(Ma1 and Ma2) were tested on Flordaguard. In June 2016, Flordaguard seedlings were

22

inoculated with 20,000 eggs and (or) J2/plant of Mf5, Ma1 and Ma2, and replicated eight

times.

In each experiment, tomato cv. Agriset 334 was used as a susceptible control to

determine inoculum viability. Peach and tomato were watered daily and fertilized weekly.

Fungicides and insecticides were applied when necessary. Tomato was harvested 2

months after inoculation, whereas peach rootstocks were harvested 2 months after

inoculation in experiment 2 and 5 months after inoculation in both experiments carried

out in 2016.

The plant roots were removed from pots and thoroughly washed to clean away

the media and debris. All root systems were weighed, rated for galls, and egg masses.

The reproductive factor (RF) and eggs per g fresh root weight (eggs per g frw) were

also determined. In experiments 3 and 4, the number of eggs per egg mass was

determined. Before ratings were made, individual root systems were soaked with 20%

(v/v) solution of Chef’s Quality food coloring (RD/Jet, LLC, College Point, NY) for 20 min

to stain egg masses (Thies et al., 2002). Gall and egg mass indices were rated based

on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 =

31-100, 5 = > 100 galls or egg masses (Taylor and Sasser, 1978). Eggs were extracted

from each root system with 1% NaOCl (Boneti and Ferraz, 1981). The RF was

calculated based on the final population per initial inoculum (Sasser et al., 1984). Eggs

per g frw for each plant were determined by counting eggs extracted from each root

system.

Five egg masses were hand-picked from each root system to determine the

number of eggs per egg mass for each isolate. Each egg mass was placed in a 1.5 ml

23

microcentrifuge tube containing 100 µl of a 2% NaOCl solution. The eggs were

loosened from the gelatinous matrix by teasing with a needle. After adding an additional

600 µl of 2% NaOCl solution, the mixture was vortexed for 2 min. Water (900 µl) was

added into the microcentrifuge tube. The suspension was mixed thoroughly and

transferred to a counting chamber. An additional 400 µl of water was used to rinse the

tube and then transferred to a 2-ml counting chamber for determining the number of

eggs under a stereo-microscope at ×400.

Data and Statistical Analysis

Variables subjected to analyses were gall and egg mass indices, reproduction

factor, the number of eggs per g fresh roots, and eggs per egg mass. Transformation of

data were made before analyses as follows: RF by fourth-root (Taylor et al., 1991); and

eggs per gram fresh root weight by arcsin(√x). Transformed data were subjected to

analysis of variance (ANOVA) using R version 3.3.0 (R Core Team, 2014) with RStudio

version 0.99.903 (RStudio, 2014), and mean separations were determined using

Tukey’s honest significant difference (HSD) (P ≤ 0.05). Unless otherwise noted, all

statistical differences were significant at the 0.05 probability level.

Field Experiment

Site Locations

Two peach orchards (Pampered Peach, LLC [PAP], Polk Co. and Capron Trail

[CT], St. Lucie Co., FL.) were chosen for determining population densities at different

soil depths over a 17-month period.

Depth Sampling

Soil and roots were collected from Pampered Peach (PAP) and Capron Trail

Farms (CT) from April 2015 to July 2016 as follows: April, June, August, and December

24

2015, March, April, and July 2016. At each orchard, four different root-knot nematode

infected trees were selected based on visual inspection. Galled roots and soil depth

samples were collected from three sites around each tree. First, a shovel was used to

collect galled roots from the top 30 cm depth, and this was followed by collecting soil

depth samples with a 10-cm-diam. bucket auger. Depths sampled at PAP were 0 to 30

cm, 31 to 60 cm, 61 to 90 cm, and 91 to120 cm deep below ground. At CT, soil depths

sampled were 0 to 30 cm, and 31 to 60 cm deep. Depth sampling at the latter was

limited to top 60 cm because of high water table.

Each soil sample was mixed thoroughly and placed in a 3.8-L sampling bag. The

samples were stored at 10 °C until processed (Barker and Nusbaum, 1968). Root-knot

nematode females from galled peach roots were hand-picked and subjected to

polyacrylamide gel electrophoresis (PAGE) for identification using esterase and malate

dehydrogenase. Nematodes were extracted from 200-cm3 of soil by centrifugal-flotation

method (Jenkins, 1964). The numbers of nematodes in the soil and root-knot nematode

J2 and eggs extracted from the roots were counted with the aid of an inverted

microscope at ×400. Other plant-parasitic nematodes extracted from soil were identified

based on their morphology. Root samples were weighed before extracting eggs and

then subjected to treatment with a 1% NaOCl solution and collected on a sieve with 25-

µm openings.

Soil Analysis

Soil was collected from the A horizon (30 cm deep) at each farm site and dried

for 2 weeks before submitting to the UF/IFAS Analytical Services Laboratory to

determine nutrient elements, pH, and percentage of organic matter. A subsample of 100

25

grams was subjected to analysis by Bouyoucos Hydrometer Method (Bouyoucos, 1951)

to determine the soil physical texture.

Data and Statistical Analysis

Variables analyzed were the number of each species of plant-parasitic

nematodes per 200-cm3 soil, and the number of root-knot nematode J2 and eggs per

gram fresh root. Data were subjected to analyses by using R version 3.3.0 (R Core

Team, 2014) with RStudio version 0.99.903 (RStudio, 2014). All results reported unless

otherwise noted were significant at the 0.05 probability level.

26

Table 2-1. The origin of root-knot nematode isolates used in this study.

Meloidogyne spp. Isolate number1 Reference

number Collection date

Origin

M. floridensis Mf1 N05-227-17B1 Before 2010 Tomato – Seminole Co., FL.

Mf2 N03-1582-2B1 Before 2010 Tomato – Indian River Co., FL.

Mf3 N04-627-5B1 Before 2010 Cucumber – Hendry Co., FL

Mf42 N03-18941 Before 2010 Peach – University of Florida Fruit Teaching Orchard, Alachua Co., FL.

Mf5 PS-4 2014 Peach – University of Florida Fruit Teaching Orchard, Alachua Co., FL.

M. arenaria Ma1 PS-1567 2014 Peach – Pampered Peach Farm, Polk Co., FL. Ma2 PS-19 2014 Peach – Rafool Farm, Polk Co., FL. M. javanica Mj1 PS-69 2014 Peach – Terry’s Farm, Pesco Co., FL. Mj2 IFC-1 2015 Peach – University of Florida Fruit Teaching Orchard,

Alachua Co., FL. M. incognita Mi1 Mi-1 2014 Cotton – Attapulgus, Decatur Co., GA Mi2 Mi-2 2014 Weed – Alachua Co., FL. 1 Reference numbers provided by the Division of Plant Industry, Florida Department of Agriculture, Gainesville, FL. 2 Mf4 is the topotype of M. floridensis.

27

CHAPTER 3 RESULTS AND DISCUSSION

Greenhouse Experiment

Host Differential Test

All five M. floridensis isolates infected and reproduced on cotton, pepper,

watermelon, and tomato, but were negative on tobacco and peanut (Table 3-1). When

repeated, Mf2, Mf4 and Mf5 failed to reproduce on pepper, whereas Mf5 failed to

reproduce on cotton. Tomato and watermelon were reported as host to M. floridensis,

and peanut was a nonhost (Handoo et al., 2004; Stanley et al., 2009; Charchar et al.,

2010). In this study, M. floridensis reproduced on cotton, pepper and tobacco, however

depending on the test, the results were variable.

Both M. arenaria isolates infected and reproduced on tobacco, watermelon, and

tomato, but not on cotton or peanut (Table 3-1). Ma2 reproduced on pepper in two trials,

however Ma1 reproduced on pepper in only one of the two tests. Based on the fact that

both M. arenaria isolates infected pepper but not peanut, they were identified as race 3

(Fargette, 1987). Three host races have been reported for M. arenaria based on host

differential tests (Fargette, 1987; Robertson et al., 2006). Peanut and pepper were

reported to distinguish these three races.

Experiment 1

Three out of four M. floridensis and two M. arenaria isolates infected Lovell

(Table 3-2). Three of four M. floridensis isolates and the two M. arenaria isolates failed

to infect Okinawa and Flordaguard. Mf1 isolate reproduced at a very low level on

Flordaguard. The two M. javanica and two M. incognita isolates induced root galling and

produced egg masses on Lovell, but they failed to infect Okinawa and Flordaguard

28

(Table 3-3). Both rootstocks have been reported as resistant to M. javanica and M.

incognita (Sharpe, 1969; Sherman et al., 1991). However, both M. floridensis and M.

arenaria isolates reproduced at a very low level on Lovell, which was the susceptible

control. In this test the plants were grown in plastic pot in a plastic covered house

without a cooling system during the heat of summer. Several plants died and those that

survived produced a small root system. It is suspected that much of the inoculum may

have become nonviable, or its infectivity was greatly reduced. Similar conditions have

been reported (Luzzi et al., 1987).

Experiment 2

The M. floridensis isolate Mf5 induced galls and reproduced on all three peach

rootstocks Flordaglo, Okinawa, and Flordaguard (Table 3-4). There was a greater

number of galls and egg masses on Flordaglo and Flordaguard than on Okinawa at the

inoculum densities of 2,000 and 10,000 (Figure 3-1, and 3-2). There was an interaction

between inoculum levels and peach cultivars for reproductive factor and eggs per gram

frw (P < 0.001, Table 3-5, Figure 3-3, and 3-4). The reproductive factor for Okinawa was

only less than the two other rootstocks. Eggs per gram frw from Flordaguard was higher

when the inocula level was 10,000 eggs and (or) J2 than when inoculated with 2,000

and 5,000 eggs and (or) J2. No difference in eggs per gram frw on Okinawa was found

regardless of inoculum level. The highest eggs per gram frw was found for Flordaglo

when inoculated with 5,000 eggs and (or) J2, followed by Flordaguard inoculated with

10,000 eggs and (or) J2 (323 and 246 for Flordaglo and Flordaguard, respectively). The

reproductive factor (RF) followed the same trend as the eggs per gram frw, except for

Flordaguard. The RF on Flordaguard were highest and lowest when the inoculum level

was 10,000 and 2,000 eggs and (or) J2, respectively, whereas the RF with 5,000 eggs

29

and (or) J2 for Flordaguard was not different from either 2,000 nor 10,000 eggs and (or)

J2/plant.

Flordaguard was identified with higher infection when the inoculum density

increased to 5,000 and 10,000 eggs and (or) J2/plant, which agreed with previous

studies (Di Vito et al., 2005; López-Pérez et al., 2006). Based on the host classification

method proposed by Sasser et al., 1984, Flordaguard would be considered as an

efficient host to M. floridensis isolate Mf5. This isolate was recently collected from peach

in the field and increased on tomato in the greenhouse for ca. 1 year before placed back

on peach.

Okinawa was found infected with M. floridensis in the field in Florida (Sharpe et

al., 1969). However, in our study, the reproduction of this nematode species on

Okinawa was at a low level and would be considered as non-host even when the

inoculum level increased to 10,000 eggs and (or) J2/ plant. Different results between

greenhouse and field tests have been reported previously (Yeates, 1982). It has been

suggested that to separate infection on resistant and susceptible cultivars a high

inoculum level should be used (Saichuk et al., 1976). This is important especially when

screening for the resistance. A high inoculation level with repeated inoculation should

be considered (Esmenjaud et al., 1992).

Experiment 3

Among the three peach rootstocks Lovell, Okinawa, and Flordaguard, Lovell was

the only susceptible to all three M. floridensis isolates (Mf1, Mf2, and Mf4) (Figures 3-5

and 3-6). Lovell was heavily infected by all three isolates and there were no differences

among the three isolates (Tables 3-6). Even Mf4, the topotype of M. floridensis, which

30

was originally collected from peach roots, failed to induce galls or reproduce on

Okinawa or Flordaguard.

Both isolates of M. arenaria infected and reproduced on Flordaguard (Figure 3-7).

Although galling was observed on Okinawa by Ma1, there was no sign of any egg

masses (Table 3-7). For the susceptible control Lovell, eggs per g frw and RF produced

by Ma1 were higher than those recovered from Flordaguard. The number of eggs per

egg mass on peach was relatively low when compared with that obtained on tomato;

Mf2 and Ma1 produced higher number of eggs per egg mass than Mf1or Mf4 (Table 3-

8).

There was a marked difference between the ability of isolates of M. floridensis to

infect and reproduce on Flordaguard. In the greenhouse, Flordaguard was only infected

by M. floridensis isolate Mf5 obtained from peach in 2014, whereas it was a nonhost to

Mf1, Mf2, and Mf4. The latter three isolates were maintained on tomato for several

years. It is possible these isolates lost their ability to infect peach after being propagated

for years on tomato (Hendy et al., 1985; Castagnone-Sereno et al., 1992; Hernandez et

al., 2004). Another explanation could be inherent variation among different isolates of M.

floridensis (Netscher and Taylor, 1979; Stirling and Cirami, 1984; Trudgill and Blok,

2001).

This is the first report of M. arenaria race 3 occurring in Florida. Also, it is the first

report that both isolates infected and reproduced on Flordaguard. M. arenaria race 3

was first reported in Europe and Uruguay where the species is known to infected

numerous crops (Fargette, 1987; Devran and Sogut, 2011; Robertson et al., 2006;

2009).

31

Okinawa was a poor host for M. arenaria race 3. Galls were observed on

Okinawa roots, but there were no egg masses found. M. arenaria may lack the ability to

complete its life cycle in Okinawa or perhaps metabolites in Okinawa roots deterred

reproduction. A similar condition was reported in Guardian roots when inoculated with M.

incognita J2 (Nyczepir et al., 1999).

Experiment 4

Both M. floridensis (Mf5) and M. arenaria (Ma1 and Ma2) infected and

reproduced on Flordaguard. All three isolates had gall and egg mass indices higher

than 3.0 (Table 3-9). There was no difference in galling, egg mass indices, or RF among

these three isolates. Mf5 and Ma2 had a RF of 0.47 and 0.39, which was the highest

and the lowest, respectively. The amount of eggs per egg mass differed among the two

nematode species. Both M. arenaria isolates produced about double the amount of

eggs per egg mass as did M. floridensis.

Our result confirmed that one isolate of M. floridensis (Mf5) and two isolates of M.

arenaria (Ma1 and Ma2) can infect and reproduce on Flordaguard.

Lower reproduction was observed of both M. floridensis and M. arenaria on

Flordaguard seedlings provided by a commercial nursery than seedlings that were

prepared from cuttings or seeds obtained from the University of Florida Fruit Teaching

Orchard, Alachua Co., FL. Lu et al. (2000) stated that medium formulation can influence

root-knot nematode infection. The commercial nursery provided seedlings that were

grown in a sand, peat and perlite media, whereas the media prepared for previously

experiments contained a combination of sand and perlite. It is possible that root-knot

nematodes reproduced poorly in the medium with peat.

32

Field Experiment

Site Description and Selection

In both PAP, Polk Co., and CT Farm, St. Lucie Co., FL., the soil was acidic and

sandy. The total percentage of silt and clay were less than 3% in all the soil depths

(Table 3-10). The soil at PAP was classified as Candler sand and the sand content

increased with depth of soil. At CT, the classification of soil was Ankona sand, and there

was a decrease in the proportion of sand with depth of soil.

At both peach orchards, root-knot (Meloidogyne spp.), ring (Mesocriconema sp.),

and lesion (Pratylenchus sp.) nematodes were found in soil collected from around the

peach rhizosphere. Based on esterase (EST) and malate dehydrogenase (MDH)

phenotype profiles obtained from single root-knot nematode females extracted from

roots collected at PAP, the root-knot nematode species was identified as M. arenaria

with EST = A3 and MDH = N3 phenotype (Figure 3-8) (Esbenshade and Triantaphyllou,

1985). This nematode species was also identified as M. arenaria race 3 based on the

host differential test (Table 3-3). The root-knot nematode extracted from peach roots at

the CT farm was identified as M. floridensis based on EST = MF3 phenotype and MDH

= N1 phenotype (Figure 3-9) (Brito et al., 2008), and based on the host differential test,

the nematode reproduced on cotton, watermelon, tomato, and produced egg masses on

pepper but no visible galls.

The ring nematodes extracted from soil collected at PAP and CT farms were

identified as Mesocriconema ornatum and M. xenoplax, respectively (Renato Inserra,

personal communication). The identities of lesion nematodes were Pratylenchus

hippeastri at PAP and P. brachyurus collected at CT (Renato Inserra, personal

33

communication). Although these parasites were found in soil, it was not established that

they parasitized peach roots.

Root-knot nematodes most frequently reported infecting peach are M. incognita,

M. javanica, and M. arenaria (Layne and Bassi, 2008). Meloidogyne hapla was also

reported to infect peach in Europe; however, the level of infection was low (Esmenjaud

et al., 1994). Some less predominant root-knot nematode species, namely M.

floridensis , M. hispanica and M. morocciensis are reported to infect peach (Sharpe et

al., 1969; Hirschmann, 1986; Rammah and Hirschmann, 1990; Esmenjaud et al., 1994).

Among ring nematodes, three species are reported infecting peach, namely M.

xenoplax, M. curvatum and M. rusticum. At least nine species of lesion nematode infect

peach worldwide (Layne and Bassi, 2008).

Population Densities of Root-Knot, Ring, and Lesion Nematodes at Pampered Peach and Capron Trail Farms.

At PAP, the number of root-knot nematode second-stage juvenile, ring and lesion

nematodes in the top 120-cm of soil was higher in 2015 than in 2016 (Figures 3-10, 3-

11, and 3-12). From April to June 2015, the number of root-knot and ring nematodes

increased in the sample collected from the 0 to 30 cm depth, with the highest numbers

occurring in June 2015 followed by a small drop in August. Root-knot nematode had

higher population densities in August 2015 at the two lower depths than at any other

sampling period. The number for root-knot and ring nematodes decreased following the

August sampling and remained consistently low at all depths. The number of lesion

nematodes recovered from soil remained relatively low over the entire sampling period

(Figure 3-12).

34

The number of eggs and J2 of root-knot nematode per g frw reached the highest

numbers in August, and was followed by the extraction of very low numbers in

December (Figures 3-16). There was an increased in numbers again in early March,

after which the numbers remained low over the next two sampling periods of April, and

July.

For the 17-month period of sampling at four soil depths (April 2015 to July 2016),

population densities of root-knot nematode J2 decreased monotonically with the lower

depths with the exception of August and December 2015. In August 2015, the highest

and the second highest population densities of root-knot nematode J2 were in the top

30 cm soil and the 61 to 90 cm-deep soil depths, respectively. In December 2015, the

trend was for the population densities to decrease and remain at relative low densities

throughout the remaining sampling periods. Ring nematodes had the same trend.

At CT, the number of root-knot nematode J2, ring, and lesion nematodes in the

top 60-cm soil fluctuated from April 2015 to July 2016 (Figures 3-13, 3-14, and 3-15).

Population densities of root-knot nematode peaked in June 2015, and again in March

2016. Ring nematode had the highest number in June 2015 and April 2016. Like the

population density of lesion nematode in PAP, CT had a low number of lesion nematode

in the soil. In the peach roots, both the numbers of root-knot nematode eggs and J2

extracted from per gram peach roots varied over the sampling period, and reached their

peak in August 2015 and again in March 2016, respectively (Figures 3-17).

For the 17-month period of sampling at CT (April 2015 to July 2016), population

densities of root-knot and ring nematodes decreased monotonically with soil depth.

Beginning in April 2015, the population densities were consistently higher in the top soil

35

depth, with only slightly lower numbers in the lower soil depth; however, this pattern

changed following the low point reached in December. Following the low numbers

recovered in December, the numbers in the lower depth remained consistently lower

than that recovered in the upper sample. Many factors can influence the distribution of

nematodes in the field. The most important biotic factor affecting their distribution in soil

is considered mainly as the root system of plants (Wallace, 1968; Norton, 1989; Norton

and Niblack, 1991). Although there is not a strong correlation between vertical

distribution of perennial or annual crop roots and the number of plant-parasitic

nematodes, some studies show that root distribution is an important factor affecting

nematode distribution in soil (Ferris et al., 1976; Ferris and Goodell, 1980; Assheuer

and Roessner, 1993; Forge et al., 1998; Verschoor et al., 2001). In this study, more

root-knot nematodes were found in the upper 30 cm soil depth, which was were most

small feeder roots of peach occurred. Few peach roots were found below the 30 cm

depth.

The seasonal distribution changes of nematodes in the soil can be influenced by

a combination of environmental factors (Yeates, 1982; Verschoor et al., 2001). An

earlier report showed that the population of root-knot nematode reached their highest

numbers in a peach orchard in August and September different than our study (Ferris et

al., 1976). In this study, the number of root-knot nematodes in the soil was relatively low

over the 17-month sampling period. However, at both sites, a higher number of eggs

and J2 were extracted from roots in August 2015 and March 2016. Since root-knot

nematodes are endoparasites, the numbers of J2 in the soil should remain relatively low,

36

especially on a perennial crop like peach in that the hatched J2 possibly return and

invade the roots (Ferris and McKenry, 1974).

37

Table 3-1. Host differential test of five isolates of Meloidogyne floridensis (Mf) and two isolates of M. arenaria (Ma)1. Isolate Differential hosts2

Test 1 Test 2

Tobacco Cotton Pepper Watermelon Peanut Tomato Tobacco Cotton Pepper Watermelon Peanut Tomato

Mf1 1.03 4.0 4.0 5.0 0.0 5.0 0.0 5.0 3.3 5.0 0.0 5.0

Mf2 1.5 2.5 4.8 5.0 0.0 5.0 0.0 4.5 1.0 5.0 0.0 5.0

Mf3 0.3 2.5 3.8 5.0 0.0 5.0 0.0 5.0 1.0 5.0 0.0 5.0 Mf4 0.0 1.8 4.0 5.0 0.0 5.0 0.0 5.0 2.8 5.0 0.0 5.0

Mf5 0.0 1.0 4.0 5.0 0.0 5.0 0.0 0.0 1.0 5.0 0.0 5.0

Ma1 3.8 0.0 0.0 5.0 0.0 5.0 1.7 0.0 4.1 3.7 0.0 5.0

Ma2 5.0 0.0 5.0 4.0 0.0 5.0 5.0 0.0 1.7 4.0 0.0 5.0 1 Mf1 - Mf5 were collected from tomato, Sanford, Seminole Co., FL., tomato, Syngenta Research Farm, Indian River Co., FL., cucumber in J and J farm, Hendry Co., FL., peach cultivar Nemaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., and peach cultivar Flordaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., respectively. Ma1, and Ma2 were collected from peach, Pampered Peach Farm, Polk Co., FL., and Rafool Farm, Polk Co., FL., respectively. 2 Tobacco (Nicotinana tabacum cv. NC95), cotton (Gossypium hirsutum cv. Deltapine 16), pepper (Capsicum annuum cv. California Wonder), watermelon (Citrullus lanatus cv. Charleston Grey), peanut (Arachis hypogaea cv. FL 07), and tomato (Solanum esculentum cv. Agriset 334). 3 The egg mass indices are based on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 =3-10, 3 = 11-30, 4 = 31-100, 5 = > 100 galls or egg masses(Taylor and Sasser, 1978).

38

Table 3-2. The reproduction of four isolates of Meloidogyne floridensis (Mf) and two isolates of M. arenaria (Ma)1 on three peach cultivars: Flordaguard, Okinawa, and Lovell.

Peach cultivar

Mf1 Mf2 Mf3 Mf4 Ma1 Ma2

GI3 EMI3 RF3 GI EMI RF GI EMI RF GI EMI RF GI EMI RF GI EMI RF Flordaguard 0.32 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Okinawa 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Lovell 0.0 0.0 0.0 3.0 3.0 0.1 1.5 1.0 0.2 1.5 2.0 0.0 3.0 3.0 0.0 3.0 2.0 0.0 1 Mf1, Mf2, Mf3 and Mf4 were collected from tomato, Sanford, Seminole Co., FL., tomato, Syngenta Research Farm, Indian River Co., FL., cucumber, Hendry Co., FL., and peach cultivar Nemaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., respectively. Ma1, and Ma2 were collected from peach, Pampered Peach Farm, Polk Co., FL., and Rafool Farm, Polk Co., FL., respectively. 2 Actual data are presented. Means are average of two or three replications. 3 Both gall (GI) and egg mass indices (EMI) were based on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100, 5 = > 100 galls or egg masses (Taylor and Sasser, 1978). RF = final population/initial population (Sasser et al., 1984). Initial population = 5,000 eggs and (or) J2/plant.

39

Table 3-3. The reproduction of two isolates of Meloidogyne javanica (Mj) and two isolates of M. incognita (Mi)1 on three peach cultivars: Flordaguard, Okinawa, and Lovell.

Peach cultivar

Mj1 Mj2 Mi1 Mi3 GI3 EMI3 RF3 GI EMI RF GI EMI RF GI EMI RF

Flordaguard 0.32 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Okinawa 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Lovell 5.0 5.0 0.75 5.0 5.0 1.1 1.5 1.0 0.2 5.0 5.0 1.0 1 Mj1, and Mj2 were collected from peach, Gulf Co., FL., and Alachua Co., FL., respectively. Mi1, and Mi2 were collected from cotton, Decatur Co., GA., and weed Alachua Co., FL., respectively. 2 Actual data are presented. Means are average of two or three replications. 3 Both gall (GI) and egg mass indices (EMI) were based on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100, 5 = > 100 galls or egg masses (Taylor and Sasser, 1978). RF = final population/initial population (Sasser et al., 1984). Initial population = 5,000 eggs and (or) J2/plant.

40

Table 3-4. The effect of three inoculum levels of Meloidogyne floridensis isolate (Mf5) collected from peach cultivar Flordaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., 2014 on three peach cultivars: Flordaguard, Okinawa, and Flordaglo.

Peach cultivar

Inoculum levels 2,000 eggs and (or) J2/plant 5,000 eggs and (or) J2/plant 10,000 eggs and (or) J2/plant

GI2 EMI2 E/g frw2 RF2 GI EMI E/g frw RF GI EMI E/g frw RF

Flordaguard 3.01a3 2.8 a 3 a 0.15 a 5.0 a 4.0 a 38 b 0.56 b 5.0 a 4.3 a 246 a 1.67 a Okinawa 1.5 b 1.3 b 14 a 0.46 a 3.0 b 1.8 b 12 b 0.16 b 2.8 b 1.0 b 8 b 0.06 b

Flordaglo 2.8 ab 2.8 a 9 a 0.46 a 5.0 a 5.0 a 323 a 3.66 a 5.0 a 4.3 a 78 a 0.74 a Tomato4 5.0 5.0 3,352 80.72 5.0 5.0 7,871 75.49 5.0 5.0 9,625 30.52 1 Means are an average of four replications. RF data was transformed by 4√x and E/g frw data was subjected to transformation by arcsin(√x) before analysis. Actual data are presented. 2 Both gall (GI) and egg mass indices (EMI) were based on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100, 5 = > 100 galls or egg masses (Taylor and Sasser, 1978). RF = final population/initial population (Sasser et al., 1984). E/g frw = eggs per g fresh root weight. 3 Means in the same column followed by the same lower case letter were not significantly different based on Tukey’s honest difference test (P ≤ 0.05) and are to be compared vertically across peach cultivars within the corresponding index of one isolate. 4 Tomato was included to ensure inocula viability.

41

Table 3-5. Results of the main effects ANOVA for the of three inocula levels of Meloidogyne floridensis isolate Mf51 on three peach cultivars: Flordaglo, Okinawa, and Flordaguard.

Source of Variation P-value

GI2 EMI2 E/g frw2 RF2

Peach cultivars <0.0001 <0.0001 0.0002 <0.0001 Inoculum levels 0.0001 0.0387 0.0002 0.0042 Peach cultivars x inoculum levels 0.9795 0.4377 0.0001 0.0003 1 Mf5 was collected from the peach cultivar Flordaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., 2014. 2 GI = gall index. EMI = egg mass index. E/g frw = eggs per g fresh root weight. RF = reproductive factor.

42

Table 3-6. The reproduction of three isolates of Meloidogyne floridensis (Mf)1 on three peach cultivars: Flordaguard, Okinawa, and Lovell.

Peach cultivar

Mf1 Mf2 Mf4

GI3 EMI3 E/g frw3 RF3 GI EMI E/g frw RF GI EMI E/g frw RF

Flordaguard 0.02 0.0 0 0.0 0.0 0.0 0 0.0 0.0 0.0 0 0.0 Okinawa 0.0 0.0 0 0.0 0.0 0.0 0 0.0 0.0 0.0 0 0.0 Lovell 5.0 a4 5.0 a 1,244 a 11.8 a 5.0 5.0 1,604 a 18.0 a 5.0 a 5.0 a 1,691 a 14.5 a

Tomato5 5.0 5.0 5,932 42.3 5.0 5.0 7,548 62.7 5.0 5.0 8,915 59.1 1 Mf1, Mf2, and Mf4 were collected from tomato, Sanford, Seminole Co., FL., tomato, Syngenta Research Farm, Indian River Co., FL., and peach cultivar Nemaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., respectively, and had been grown on tomato for at least 5 years. 2 Means are an average of four replications. RF data was transformed by 4√x and E/g frw data was subjected to transformation by arcsin(√x) before analysis. Actual data are presented. 3 Both gall (GI) and egg mass indices (EMI) were based on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 =11-30, 4 = 31-100, 5 = >100 galls or egg masses (Taylor and Sasser, 1978). RF = final population per initial population (Sasser et al., 1984). Initial population = 10,000 root-knot nematode eggs and (or) second-stage juveniles. E/g frw = eggs per g fresh root weight. 4 Means in the same row followed by the same lower case letter were not significantly different based on Tukey’s honest difference test (P ≤ 0.05) and are to be compared vertically across peach cultivars within the corresponding index of one isolate. 5 Tomato was included to ensure inocula viability.

43

Table 3-7. The reproduction of Meloidogyne arenaria isolates Ma1 on three peach cultivars: Lovell, Okinawa, and Flordaguard and Ma21 on peach cultivar Flordaguard.

Peach cultivar Ma1 Ma2

GI3 EMI3 E/g frw3 RF3 GI EMI E/g frw RF

Flordaguard 2.92b4 3.5 b 377 b 3.6 b 5.0 5.0 259 3.4 Okinawa 2.6 b 0.0 c 0 c 0.0 c NA6 NA NA NA Lovell 5.0 a 5.0 a 1,485 a 18.0 a NA NA NA NA Tomato5 5.0 5.0 8,742 42.4 NA NA NA NA 1 Ma1 and Ma2 were collected from peach, Pampered Peach Farm, Polk Co., FL., and peach,

Rafool Farm, Polk Co., FL., respectively. 2Means are an average of four replications. RF data was transformed by 4√x and E/g frw data was subjected to transformation by arcsin(√x) before analysis. Actual data are presented. 3 Both gall (GI) and egg mass indices (EMI) were based on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 =11-30, 4 = 31-100, 5 = >100 galls and egg masses (Taylor and Sasser, 1978). E/g frw = eggs per g fresh root weight. RF = final population per initial population (Sasser et al., 1984). Initial population = 10,000 eggs and (or) second-stage J2/plant. 4 Means in the same column followed by the same lower case letter were not different based on Tukey’s honest significant difference test (P ≤ 0.05) and are to be compared vertically across peach cultivars within the corresponding index of one isolate. 5 Tomato was included to ensure inocula viability. 6 NA = plants were not inoculated due to lack of inoculum.

44

Table 3-8. Number of eggs per egg mass and range produced by Meloidogyne spp.1 on peach rootstock cvs. Flordaguard and Lovell.

Peach cultivar Mf1 Mf2 Mf4 Ma1 Ma2

Flordaguard Ni6 Ni Ni 65 Ab4 (0-117) 71 A (51-89) Lovell 732 B3 (35-151) 97 A (45-189) 58 B (25-103) 92 Aa (76-105) NA6 Tomato5 455(219-568) 141 (93-193) 248 (125-382) 267 (75-405) NA 1 Mf1, Mf2, and Mf4 were collected from tomato, Sanford, Seminole Co., FL., tomato, Syngenta

Research Farm, Indian River Co., FL., and peach cultivar Nemaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., respectively. Both Ma1 and Ma2 were obtained from peach grown in Polk Co., FL. 2 Means were an average of seven replications of five egg masses per plant. Data was

transformed arcsin(√x) before analysis. Actual data are presented. 3 Means in the same row followed by the same upper case letter were not different based on Tukey’s honest significant difference test (P ≤ 0.05) and are to be compared horizontally across isolate within the corresponding index of one peach cultivar. 4 Means in the same column followed by the same lower case letter were not different based on Tukey’s honest significant difference test (P ≤ 0.05) and are to be compared vertically across peach cultivars within the corresponding index of one isolate 5 Tomato was included to ensure inocula viability. 6 NA = plants were not inoculated due to lack of inoculum. Ni = no egg mass was found on the peach roots.

45

Table 3-9. The reproduction of one isolates of Meloidogyne floridensis and two isolates of Meloidogyne arenaria1 on peach cultivar Flordaguard.

Root-knot nematode isolate GI3 EMI3 E/g frw3 RF3 E/EM (Range) 3

Mf5 3.92 A4 3.8 A 58 A 0.46 A 32 B (10,72) Ma1 3.1 A 3.1 A 65 A 0.40 A 59 A (31,93) Ma2 3.0 A 3.1 A 60 A 0.39 A 69 A (45,120)

1 Mf5 was collected from Flordaguard, Fruit Teaching Orchard, University of Florida, Gainesville, FL., and both Ma1 and Ma2 were obtained from peach grown in Polk Co., FL. 2 Means are an average of eight replications. RF data was transformed by 4formed E per g frw

data and E/EM were subjected to transformation by arcsin(√x) befor analysis. Actual data are presented. 3 Both gall (GI) and egg mass (EMI) indices were based on a 0 to 5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 =11-30, 4 = 31-100, 5 = >100 galls or egg masses (Taylor and Sasser, 1978). RF = final population per initial population (Sasser et al., 1984). Initial population = 20,000 eggs and (or) second-stage juveniles. E/g frw = eggs per g fresh root weight. E/EM = eggs per egg mass. 4 Means in the same row followed by the same upper case letter were not significantly different based on Tukey’s honest difference test (P ≤ 0.05) and are to be compared horizontally across isolates within the corresponding index of one peach cultivar.

46

Table 3-10. Soil characteristics of the soil depths sampled at Pampered Peach and Capron Trial Farms.

Peach orchard

Soil depth (cm)

Soil texture1 OM (%)2

pH

Elements3

Sand (%)

Silt (%)

Clay (%)

P (mg/kg)

K (mg/kg)

Ca (mg/kg)

Zn (mg/kg)

Cu (mg/kg)

Na (mg/kg)

TKN4 (mg/kg)

PAP, Polk Co., FL.

0-30 96.6 0.9 2.5 0.93 6.02 27.40 53.88 923.8 8.72 4.08 51.89 390.1

30-60 97.8 1.5 0.7 0.47 6.38 8.08 33.01 347.5 3.06 0.78 31.79 107.3

60-90 98.8 0.2 1.0 0.33 6.42 18.55 23.93 219.5 2.00 0.58 23.95 52.68

90-120 99.3 0.2 0.5 0.20 6.43 38.20 26.01 288.8 2.59 0.45 20.20 67.46

CT, St. Lucie

Co., FL. 0-30 98.5 1.0 0.5 2.27 5.49 133.2 23.60 442.3 39.20 74.56 7.94 761.2

30-60 97.5 1.3 1.2 1.60 4.26 127.6 20.68 251.5 10.67 35.05 7.52 426.5 1 Soil texture was measured by Bouyoucos Hydrometer Method (Bouyoucos, 1951). 2 OM = Organic matters content. 3 Elements in dry soil samples were tested in UF/IFAS Analytical Services Laboratories. 4 TKN = total Kjeldahl Nitrogen.

47

Figure 3-1. Gall index responses of inoculum levels to peach rootstock cultivars.

0

1

2

3

4

5

6

Flordaguard Lovell Flordaglo

Gal

l In

de

x

2,000 eggs and (or) J2/plant



48

Figure 3-2. Egg mass index responses of inoculum levels to peach rootstock cultivars.

0

1

2

3

4

5

6


Egg

Mas

s In

de

x




49

Figure 3-3. Number of root-knot nematode eggs per gram fresh root weight responses of inoculum levels to peach

rootstock cultivars.

0

50

100

150

200

250

300

350

400


Nu

mb

er

of

roo

t-kn

ot

ne

mat

od

e e

ggs

pe

r gr

am f

resh

ro

ot

we

igh

t




50

Figure 3-4. Reproductive factor responses of inoculum levels to peach rootstock cultivars.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


Re

pro

du

ctiv

e F

acto

r




51

Figure 3-5. Galling on peach rootstock cv. Lovell roots caused by Meloidogyne

floridensis isolate Mf2, that was originally collected from tomato grown at the Syngenta Research Farm, Indian River Co., FL. A) Photo viewed from the top of the whole root system. B) Photo viewed from the top one-half of the root system. Photos courtesy of author.

B.

A.

52

Figure 3-6. Galling on peach rootstock cv. Lovell roots caused by Meloidogyne

floridensis isolate Mf4 (topotype), that was originally collected from peach rootstock cv. Nemaguard at the Peach Research Orchard, the University of Florida, Alachua Co., FL. A) Photo viewed from the top of the whole root system. B) Photo viewed from the top one-half of the root system. Photos courtesy of author.

B.

A.

53

B.

A.

Figure 3-7. Galling on peach rootstock cv. Flordaguard roots caused by Meloidogyne

arenaria isolate Ma2, that was originally collected from peach at the Rafool Farm, Polk Co., FL. A) Photo viewed from the top of the whole root system. B) Photo viewed from the one top half of the root system of Flordaguard. Photos courtesy of author.

54

Figure 3-8. Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne arenaria females extracted from roots of peach collected from Pampered Peach Farm, Polk Co., FL. Lane 1 and lane 14 = single female of M. javanica as control (MDH = N1 and EST = J3). Lane 2-13 and lane 15 = single female of M. arenaria (MDH= N3 and EST = A2). Photo courtesy of author.

Figure 3-9. Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne floridensis females extracted from roots of peach collected from Capron Trail Farm, St. Lucie Co., FL. Lane 1 and lane 14 = single female of M. javanica as control (MDH = N1 and EST = J3). Lane 2-13 and lane 15 = single female of M. floridensis (MDH = N1 and EST = MF3). Photo courtesy of author.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MDH

EST

MDH

EST

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MDH

EST

MDH

EST

55

Figure 3-10. Seasonal changes in the mean number of Meloidogyne arenaria second-stage juveniles (J2) extracted from

200 cm3 of soil collected from four soil depths at the Pampered Peach Farm, Polk Co. FL. Each data point was the mean of 12 samples.

0

20

40

60

80

100

120

17 Apr 2015 17 Jun 2015 19 Aug 2015 14 Dec 2015 1 Mar 2016 11 Apr 2016 14 Jul 2016

Nu

mb

er

of

roo

t-kn

ot

ne

mat

od

e J

2/2

00

cm

3 o

f so

il

Date

0 - 30 cm below ground




56

Figure 3-11. Seasonal changes in the mean number of Mesocriconema ornatum extracted from 200 cm3 of soil collected

from four soil depths at the Pampered Peach Farm, Polk Co. FL. Each data point was the mean of 12 samples.

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150


Nu

mb

er

of

rin

g n

em

ato

de

/20

0 c

m3

of

soil

Date





57

Figure 3-12. Seasonal changes in the mean number of Pratylenchus hippeastri extracted from 200 cm3 of soil collected from four soil depths at the Pampered Peach Farm, Polk Co. FL. Each data point was the mean of 12 samples.

0

5

10

15

20


Nu

mb

er

of

lesi

on

ne

mat

od

e /

20

0 c

m3

of

soil

Date





58

Figure 3-13. Seasonal changes in the mean number of Meloidogyne floridensis second-stage juveniles (J2) extracted from 200 cm3 of soil collected from two soil depths at the Capron Trial Farm, St. Lucie Co. FL. Each data point was the mean of 12 samples.

0

5

10

15

20

25


Nu

mb

er

of

roo

t-kn

ot

ne

mat

od

e J

2/2

00

cm

3 o

f so

il

Date



59

Figure 3-14. Seasonal changes in the mean number of ring nematodes Mesocriconema xenoplax extracted from 200 cm3

of soil collected from two soil depths at the Capron Trial Farm, St. Lucie Co. FL. Each data point was the mean of 12 samples.

0

10

20

30

40

50

60

70


Nu

mb

er

of

rin

g n

em

ato

de

/20

0 c

m3

of

soil

Date

0 - 30cm below ground

31 - 60cm below ground

60

Figure 3-15. Seasonal changes in the mean number of Pratylenchus brachyurus extracted from 200 cm3 of soil collected

from two soil depths at the Capron Trial Farm, St. Lucie Co. FL. Each data point was the mean of 12 samples.

0

2

4

6

8

10

12


Nu

mb

er

of

lesi

on

ne

mat

od

e/2

00

cm

3o

f so

il

Date



61

Figure 3-16. Seasonal changes in the mean number of root-knot nematode eggs and root-knot nematode second-stage

juveniles (RKN J2) extracted per gram of peach roots collected from Pampered Peach Farm, Polk Co. FL. Each data point was the mean of 12 samples.

0

500

1000

1500

2000

2500

3000

3500

4000

0

50

100

150

200

250

300

350

400

17 Jun 2015 19 Aug 2015 14 Dec 2015 1 Mar 2016 11 Apr 2016 14 Jul 2016

Nu

mb

er

of

roo

t-kn

ot

ne

mat

od

e e

ggs/

g fr

esh

ro

ots

Nu

mb

er

of

roo

t-kn

ot

ne

mat

od

e J

2/g

fre

sh r

oo

ts

Date

RKN J2

Eggs

62

Figure 3-17. Seasonal changes in the mean number of root-knot nematode eggs and root-knot nematode second-stage

juveniles (RKN J2) extracted per gram of peach roots collected from Capron Trial Farm, St. Lucie Co. FL. Each data point was the mean of 12 samples.

0

500

1000

1500

2000

2500

3000

0

25

50

75

100

17 Jun 2015 19 Aug 2015 14 Dec 2015 1 Mar 2016 11 Apr 2016 14 Jul 2016

Nu

mb

er

of

roo

t-kn

ot

ne

mat

od

e e

ggs/

g fr

esh

ro

ots

Nu

mb

er

of

roo

t-kn

ot

ne

mat

od

e J

2/g

fre

sh r

oo

ts

Date

RKN J2

Eggs

63

APPENDIX ISOENZYME ELETROPHORENSIS FOR THE IDENTIFICATION OF Meoidogyne spp.

Figure A-1. Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne arenaria females extracted from tobacco roots. Ma1 was collected from peach at Pampered Peach Farm, Polk Co., FL. Lane 2 and lane 15 = single female of M. javanica as control (MDH = N1 and EST = J3). Lane 1 and lane 2-14 = single female of M. arenaria (MDH= N3 and EST = A2). Photo courtesy of author.

Figure A-2. Isozyme profiles from malate dehydrogenase (MDH) and esterase (EST) of Meloidogyne arenaria females extracted from pepper roots. Ma2 was collected from peach at Rafool Farm, Polk Co., FL. Lane 1 and lane 14 = single female of M. javanica as control (MDH = N1 and EST = J3). Lane 2-13 and lane 15 = single female of M. arenaria (MDH= N3 and EST = A2). Photo courtesy of author.

MDH

EST

MDH

EST

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MDH

EST

MDH

EST

64

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BIOGRAPHICAL SKETCH

Sai Qiu was born in 1987, in Zhengzhou City, Henan Province, China. In 2010,

she graduated from Zhengzhou University of Light Industry and earned a Bachelor of

Science degree in biotechnology. During the four-year’s university life, she started to be

interested in plants. From 2010 to 2013, she studied viticulture in Northeast Agricultural

and Forestry University. These three years gave her a broad view of plant and plant

disease. In 2014, she began her Master of Science degree in Entomology and

Nematology Department, University of Florida to study the root-knot nematodes on

peach.

evaluation of root-knot nematodes (meloidogyne...

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