Academic Year: 2015-2016

Thesis submitted to obtain the degree of Master of Science in Nematology

Host plant status of different green manure plants for Pratylenchus

penetrans and Meloidogyne chitwoodi

Alexander Mbiro

Promoter and supervisor: Prof. Dr. Ir. Wim Wesemael

GHENT UNIVERSITY. FACULTY OF SCIENCE. DEPARTMENT OF BIOLOGY.

Host plant status of different green manure plants for

Pratylenchus penetrans and Meloidogyne chitwoodi

Alexander MBIRO1, Wim .M.L.WESEMAEL1,2

1Ghent University, Department of Biology, K.L. Ledeganckstraat 35, 9000, Gent, Belgium

2Institute for Agricultural and Fisheries Research (ILVO), Burgemeester Van Gansberghelaan

96, 9820 Merelbeke, Belgium

Declaration

Submitting in this thesis, I declare that this work has never been submitted either in a whole or

part to this or any other institution of higher learning for any other degree and is, except where

otherwise stated, the original work of the author.

Abstract

Nine different green manure crops (different species or cultivars) were evaluated for their

potential use in management of Pratylenchus penetrans and Meloidogyne chitwoodi. Firstly, a

resistance screening test for each cultivar was carried out in small yellow tubes filled with soil

and inoculated with 100 P. penetrans (juveniles and adults) or M. chitwoodi (second-stage

juveniles). Eight weeks after inoculation, each cultivar was assessed for its resistance or

susceptibility to P. penetrans and M. chitwoodi. Based on the reproductive factor, bird’s-foot

trefoil cv. Franco, English ryegrass cv. Meltador, arugula cv. Nemat and fodder radish line

RsV79/80 were resistant to P. penetrans. For M. chitwoodi, Alfalfa cv. Alpha, bird’s-foot trefoil

cv. Lotar, bird’s-foot trefoil cv. Bull and fodder radish line RsV79/80 showed less than one egg

mass per root system being formed eight weeks after inoculation. These cultivars showed a high

level of resistance to M. chitwoodi multiplication. Secondly, a host evaluation pot test was

carried out for five green manure plants either singly or a mixture of cultivars, each inoculated

with 500 P. penetrans (juveniles and adults) or M. chitwoodi (second-stage juveniles). Each

cultivar or mixture was harvested 8 weeks after inoculation. Nematodes were extracted from

both roots and soil to assess the final nematode population. Fodder radish line RsV79/80, arugula

cv. Nemat and arugula- fodder radish mixture were non to poor hosts to both P. penetrans and

M. chitwoodi. Based on our results selected green manure crops or mixtures of green manure

crops can be used to control both P. penetrans and M. chitwoodi.

Key words: Plant-parasitic nematodes, resistance, susceptibility, reproductive factor.

1

INTRODUCTION

Plant-parasitic nematodes (PPN) do feed, reproduce on living plants and are capable of active

migration in the rhizosphere, on aerial plant parts and inside the plant especially the root (Dong

& Zhang, 2006). Decraemer et al. (2013), estimates that there are over 4000 species of plant-

parasitic nematodes described to date and they cause an important constraint on the agricultural

crop production globally. Economic loss caused by plant-parasitic nematodes was estimated to

be at $US80 billion per year by end of 2010 (Nicol et al., 2011). This figure is most likely to be

an underestimation, as most agricultural farmers in tropics are unaware of even the existence of

nematodes due to their microscopic nature, the atypical symptoms caused and their synergistic

association with other pathogens (De Waele & Elsen, 2007; Jones et al., 2013).

On a worldwide basis, root-knot nematode (Meloidogyne spp.), cyst nematode (Heterodera spp.

and Globodera spp.) and root lesion nematode (Pratylenchus spp.) are the first three in their

respective order of the ten most important and common genera of plant-parasitic nematodes

(Jones et al., 2013). The sedentary endoparasitic nematodes (Globodera, Heterodera,

Meloidogyne) (Back et al., 2002), semi-endoparasitic nematode (Rotylenchulus) and migratory

endoparasitic nematodes (Pratylenchus, Ditylenchus, Bursaphelenchus, Aphelenchoides and

Anguina) are the genera most commonly reported to be involved in disease complexes with

fungal and bacterial pathogens (Back et al., 2002; Moens & Perry, 2009).

As of October 2015, a total of 101 root-knot nematode species (Meloidogyne spp.) have been

described (Wesemael pers.comm). The most economically important species of Meloidogyne in

cooler climates are; M. naasi, M. hapla, M. chitwoodi and M. fallax, while M. arenaria, M.

javanica and M. incognita are the most common species in warmer conditions of southern

Europe (Moens & Perry, 2009; Wesemael et al., 2011). Meloidogyne chitwoodi and M. fallax are

the two most important species in Europe because they are quarantine pests (De Waele & Elsen,

2007; Wesemael et al., 2011).

Meloidogyne chitwoodi can parasitize a wide range of host plants which can be classified as

good hosts, maintenance hosts, poor hosts or non-hosts depending on host suitability for

nematode reproduction (Ferris et al., 1993). The classification may also vary with the nematode

ability to adapt to a particular environmental condition and the management system used.

2

Some of M. chitwoodi hosts are crop plants of economic importance, green manure plants (Cherr

et al., 2006) and common weed species (Kutywayo & Been, 2006). The common excellent crop

hosts include; potato (Solanum tuberosum), carrot (Daucus carota) and tomato (Solanum

lycopersicum) (Ferris et al., 1993). Barley (Hordeum vulgare), maize (Zea mays), oats (Avena

sativa), sugarbeet (Beta vulgaris var. saccharifera), wheat (Triticum aestivum) are maintenance

hosts (Ferris et al., 1993) while poor to non-host plants include; amaranth, oilseed radish, oilseed

rape, and safflower (Ferris et al., 1993). As for the green manure crops; a number of cultivars of

oil radish are known to be maintenance hosts while buckwheat (Fagopyrum esculentum),

rapeseed (Brassica napus), sundangrass (Sorghum vulgare), horsebean (Canavalia ensiformis),

velvetbean (Mucuna deeringina) castor (Ricinus communis), showy crotalaria (Crotalaria

spectabilis), joint-vetch Aeschynomene Americana), marigolds (Tagetes minuta and T. erecta),

sesame (Sesamum indicum cv. Paloma), barley (H. vulgare), are known to be in the range of poor

to non-host plants (Al-Rehiayani & Hafez, 1998). However, studies indicate that rapeseed as

green manure crop significantly reduces potato damage caused by M. chitwoodi (Mojtahedi et

al., 1993)

Genus Pratylenchus differ from root-knot nematode (RKN) in that they enter and leave root

tissues during their life cycle, move actively through soil and penetrate the root tissues for

feeding and reproduction (Esteves et al., 2015). Reduced growth, occasional yellowing of the

foliage and severe necrosis in roots and tubers are the major symptoms associated with the

nematodes of Pratylenchus (Castillo & Vovlas, 2007). With over 70 species of Pratylenchus

(Duncan et al., 2013), the most important species in agriculture are; P. crenatus, P. neglectus, P.

penetrans, P. thornei, P. brachyurus, P. coffeae (Jones et al., 2013). As potato is a good host of

Pratylenchus, P. penetrans was the most abundant species followed by P. neglectus, P. crenatus

and lastly P. thornei in Portugal (Esteves et al., 2015).

The migratory endoparasitic nematode P. penetrans has a wide host range, with over 350 host

plant species recorded (Mizukubo & Adachi, 1997; Moens & Perry, 2009; Duncan et al., 2013).

Among them are cultivated crops, fruits, vegetables, green manure crops and numerous weeds.

Thus this makes the species difficult to manage with crop rotation (Jensen, 1953; Townshend &

Davidson, 1960; Manuel et al., 1980). The common cultivated crops attacked by P. penetrans

are; apple, cherry, citrus, roses, tomato, potato, corn, sugarbeet, ornamentals like Narcissus spp.

3

(Slootweg, 1956; Duncan et al., 2013). The seemingly good host green manure crops includes;

kura clover (Trifolium ambiguum), alsike clover (Trifolium hybridum), white clover (Trifolium

repens), oat (Avena sativa), and rye (Secale cereale) (Thies et al., 1995). On the other hand, P.

penetrans does not reproduce well on some green manure plants and they are termed as poor or

non-hosts. Among them are; pearl millet (Pennisetum glaucum), tall fescue (Festuca

arundinacea), perennial ryegrass (Lolium perenne), forage sorghum (Sorghum bicolor),

sudangrass (Sorghum sudanense), sweetclover (Melilotus alba), crownvetch (Coronilla varia)

and MNGRN-16alfalfa (Medicago sativa) (Thies et al., 1995).

Many different control strategies are being applied in agriculture and these include: chemical,

physical, cultural, genetic (resistance) and biological control (Nicol & Rivoal, 2008).

Specifically, effective control of root-knot and lesion nematodes commonly calls for the

integrated pest management approach, including the use of crop rotations with non-host plants,

the use of resistant cultivars if available, fallow, organic amendments (Haydock et al., 2013;

Viaene et al., 2013; Kruger et al., 2015). The overall aim of using these management strategies is

to decrease the nematode population densities below damage thresholds before the next primary

host crop is cultivated (Nicol & Rivoal, 2008).

Green manure crops are crops of economic importance to the soil and crop productivity. They

have been in existence in traditional agriculture for many decades but large scale agricultural

systems did not entirely adopt their use due to efficient and cost effective use of fertilizers and

pesticides that have been readily available on the market (Viaene et al., 2013). The primary

benefits of green manure crops are to; 1) protect the soil from erosion; 2) increase soil nutrients;

3) improve soil properties such as water-holding capacity; and 4) provide an energy source for

microbes, contributing to soil activity and biodiversity (Cherr et al., 2006; Ortiz et al., 2015).

Secondarily, green manure crops are used and applied in agricultural fields in the control of

soilborne pathogens and their mechanisms of action vary with species (Ortiz et al., 2015). Green

manure treatments may play a role in disease management by changing the Streptomycete

communities in soils, leading to pathogen suppression (Wiggins & Kinkel, 2005) or resulting in

bacterial communities that may induce plant systemic resistance (Cohen et al., 2005).

Some green manure crops act as non-host or poor host to the pathogen, produce allelochemicals

that are toxic or aggressive toward pathogens or stimulate antagonists of plant parasitic

4

nematodes (Hooks et al., 2010; Ortiz et al., 2015). A good manure crop grown for the

management of plant-parasitic nematodes is characterized by having non-host or poor host status

for the target nematode and/ or being suppressive to the population growth in the soil (Viaene &

Abawi, 1998).

Additionally, though different manure crops have been proved to be a ready source of

nematicidal compounds to suppress a number of plant-parasitic nematodes, some are good hosts

as well. Within a single green manure crop species, its cultivars may vary in their suppressive

effect to a single particular nematode species. In spite of all management strategies applied, P.

penetrans and M. chitwoodi continue to be a big threat to agricultural sector in Europe as far as

crop yield is concerned. Therefore, this study screened for resistance of different green manure

plant cultivars for P. penetrans and M. chitwoodi. In addition, the study evaluated the

reproductive potentials of P. pentrans and M. chitwoodi on different green manure cultivars

planted either singly or as a mixture.

MATERIALS AND METHODS

Nematode culture

Root-knot nematode, M. chitwoodi and root-lesion nematode, P. penetrans were used throughout

the study. The population of M. chitwoodi originated from a field in Belgium and was

maintained as a pure culture at Institute for Agricultural and Fisheries Research (ILVO) on

tomato raised under greenhouse conditions (18 - 23°C, with 16:8 hours of light and darkness

respectively), in a 16 cm diameter pot size with 2 litre volume of soil. The nematodes were mass

cultured on potato tubers (Solanum tuberosum cv. Bintje) in closed containers.

Prior to planting, the potato tubers were thoroughly washed in tap water to remove soil particles

and thereafter disinfected with a 5% NaOCl solution for a maximum of 4 minutes. After

disinfection, the tubers were rinsed with tap water to remove the disinfectant (NaOCl). The

rinsed tubers were spread on soft tissue paper and left at room temperature with maximum light

for about three weeks to sprout. 200 g of sterilized white river sand was placed in each closed

plastic container (10 cm diameter and 0.5 litre volume) and 30 ml of tap water was added. One

sprouted potato tuber was placed in each closed container with sprouting roots in the soil. The

closed containers were kept in the dark for 2 weeks to allow establishment of roots. After root

5

establishment, each closed container was singly inoculated with 2000 second-stage juveniles (J2)

of M. chitwoodi. The inoculated closed containers were stored in an incubator at 20-22°C in the

dark room for 10-14 weeks to allow nematode reproduction. After reproduction, potato roots

were chopped, placed on Baermann funnel to extract nematodes (Baermann, 1917) and freshly

hatched J2 which were used for inoculation were collected after 24 hours.

The P. penetrans population originated from a maize field in Belgium and was maintained and

mass cultured as pure culture on carrot discs at ILVO. Unblemished carrots with a cylindrical

shape and fresh leaves were selected. All the working tools plus laminar flow were disinfected

with 70% ethanol. The carrots were thoroughly cleaned with distilled sterile water before

peeling. With the help of forceps, the carrots were peeled using a peeling knife, by first dipping

in 70% ethanol for a few seconds and flamed over spirit lamp. The peeling knife was

continuously moistened with 70% ethanol between peelings to avoid contamination. The peeled

carrots were cut into small discs of about 1 cm thickness with a 3-4 cm diameter using the

sterilized knife. Using the sterilized forceps one carrot disc was placed into sterile disposable

petri dishes of 5 cm diameter and sealed with parafilm. The carrot discs were then kept in the

incubator at 21°C for 3 weeks.

The petri dishes with the prepared carrot discs were removed from the incubator and placed on

the laminar flow bench. With a micropipette, a solution of nematode inoculum containing about

30 infective juveniles and few males were inoculated on top of the discs and petri dishes were

sealed with parafilm. The inoculated discs were placed in a dark container and later transferred to

an incubator maintained at a temperature of 21°C for a period of 10 weeks. Callus formation

(whitish matter) on the surface of the carrot discs was observed, indicating formation of healthy

cultures during incubation. After 10 weeks, infective juveniles and adults were extracted from

infected carrot discs with brown coloring using a modified Baermann funnel technique under a

mistifier after 24 hours. The freshly hatched mixtures of juveniles and adults were used for

inoculation of different green manure plants for the two tests.

Green manure crops and cultivars

A combination of eleven cultivars was used in this study. Eight cultivars of green manure plants

and one candivar (fodder radish, Raphanus sativus, line RsV79/80) were included in the study.

Two non-green manure plants; maize and tomato were used as positive controls in both

6

resistance screening and host evaluation tests of P. penetrans and M. chitwoodi respectively

(Table 1). Meanwhile a fallow was used as a negative control for host evaluation test for each

nematode species.

Table 1: Plant common names, scientific names seed source and 1000 grain weight.

Common name Scientific name Cultivar

1000 grain

weight (g) Seed source

Red clover Trifolium pretense Lemmon 2 ILVO, Belgium

White clover Trifolium repens Melital 1 ILVO, Belgium

Bird’s-foot trefoil Lotus corniculatus Lotar 1.3 Oseva Uni, Czech Republic

Bird’s-foot trefoil Lotus corniculatus Bull 1.5

Feldsaaten Freudenburger,

Germany

Bird’s-foot trefoil Lotus corniculatus Franco 1.1 Italy

English ryegrass Lolium multiflorum Meltador 3 ILVO, Belgium

Alfalfa Medicago sativa Alpha 2 Barenbrug, The Netherlands

Arugula Eruca sativa Nemat 1.6 Alliance, Belgium

Fodder radish Raphanus sativus RsV79/80 16.4 ILVO, Belgium

Maize Zea mays LG3220 32.6 Limagrain, Belgium

Tomato Solanum lycopersicum Marmande 1.4 AVEVE, Belgium

Resistance screening test

Resistance screening of each cultivar was carried out in small yellow tubes (RLC4 type) of 3 cm

diameter, and 16 cm height with a surface area of 7x10-4m2 made by Stuewe and Sons, USA.

One seed from each cultivar was placed in a plastic yellow tube containing soil sterilized at

100°C for 16hours. The soil comprised of 74% sand, 14% sandy loam, 6% clay, 5% loam, 1%

organic matter content and a neutral pH.

After seed germination and root establishment, each tube was inoculated with 100 J2 of M.

chitwoodi or 100 P. penetrans (a mixture of juveniles and adults). The plants were watered daily,

grown in the greenhouse at a temperature range of 18-23°C and received 16:8 hours of light and

darkness respectively. The experiment was terminated 8 weeks after nematode inoculation.

7

Plant harvest during resistance screening test

For M. chitwoodi, roots were thoroughly washed clean and separated from the soil. The roots

were then dipped in a 1 litre solution of 0.15 g phloxine B for 15 minutes to stain the gelatinous

matrix of egg masses produced by the female M. chitwoodi on the roots. After staining, root

systems were rinsed in tap water and the number of egg masses per root system were observed

and counted using a binocular microscope and thereafter quantified. The soils were not

considered and hence it was discarded after washing the roots.

For P. penetrans, roots were thoroughly washed, chopped in small pieces of about 2 cm long and

macerated in a laboratory blender (Waring commercial) for one minute. The blended roots were

added to the beaker containing soil suspension. The root soil mixture was subjected to an

automated zonal centrifuge technique (Hendrickx, 1995).

The automated zonal centrifuge technique works based on the principle of density differences;

root and soil samples are first diluted to 1 litre and half of the dilution is taken up by the machine

for extraction. The extracted sample of 500 ml is subjected to; 1), MgSO4 at a density of 1.20

kg.m-3 which plays a role in separating particles with lower and higher specific gravity than its

own specific gravity. 2), water so that nematodes are retained at the interface with the MgSO4

solution and 3) kaolin suspension which is added at the end of the centrifugation cycle to the

rotor to avoid soil particles, root and other debris from mixing with the nematode suspension

when the centrifugation process stops. At the end of the centrifugation, a supernatant of clean

water and MgSO4 containing the nematodes is collected in a small beaker of 150ml via the

hollow shaft of the rotor (figure 1). The rotor and the tubes are cleaned automatically after each

sample to avoid contamination of samples (Wander et al., 2007).

The final population (Pf) of P. penetrans for each sample was obtained by counting nematodes in

the whole 40 ml of the supernatant and multiplying by a factor of 2. These nematode final

population (Pf) included eggs, juveniles and adults from both organic (root) and mineral (soil)

fractions.

Host status evaluation test

The evaluation of host status of individual or plant mixtures was carried out in pots of 16 cm

diameter, 15.5 cm height with a 2 litre volume of soil and surface area of 0.02 m2. The number of

seeds planted in the pot was determined depending on the surface area of the pot, the seed

8

density of the plant/cultivar and whether planted individually or as a mixture. Table 2 shows the

details of the plant/cultivar in relation to seed density. White clover, red clover, English ryegrass,

fodder radish and arugula were singly planted in pots. A fallow for each nematode species was

also set up without any green manure crop planted. Two mixtures were set up for each nematode

species and these included the clover- English ryegrass mixture, and arugula- fodder radish

mixture. Seeds from each cultivar(s) were sown in pots containing sterilized soil with soil

properties as mentioned above.

Table 2: crop seed density and number of seeds per pot.

Plant

(cultivar)

seed

density/hectare

(g/10000 m2)

seed density/pot

(g/0.02 m2) No of seeds/pot

Seeding

status

Red clover 20000 0.04 20 Single

White clover 10000 0.02 20 Single

English ryegrass 30000 0.06 20 Single

Arugula 8000 0.016 10 Single

Fodder radish 40000 0.08 6 Single

Red-white clover-

ryegrass

7000,

3000,20000 0.014, 0.006,0.04 7, 6, 13 Mixture

Arugula-fodder

radish 8000, 40000 0.016, 0.08 10, 6

Mixture

After seed germination and root establishment, each pot was inoculated with an initial population

(Pi) of 500 J2 of M. chitwoodi or 500 P. penetrans (a mixture of juveniles and adults). The plants

were watered daily, grown in the greenhouse at a temperature range of 18-23°C, received 16:8

hours of light and darkness respectively. The experiment was terminated 8 weeks after nematode

inoculation. The experimental setup was a randomized complete block design with five

replicates per plant/mixture for each nematode species.

9

Nematode extraction during host status test

The organic (root) and mineral (soil) fractions were extracted separately. For both M. chitwoodi

and P. penetrans roots were thoroughly washed clean, fresh root weight taken, chopped in small

pieces of 2 cm long, homogenized and a subsample of 5 g was macerated in a laboratory blender

(Waring commercial) for one minute. The subsample of blended roots was sieved and added in a

plastic beaker. After homogenization of 2000 cm3 soil, a subsample of 200 cm3 of soil was

sieved and added in a 1 litre plastic beaker. The root and soil subsamples separately were

subjected to an automated zonal centrifuge technique (Hendrickx, 1995) as described above.

After automated zonal centrifugation, the collected nematode suspension was left to settle down

for at least 3 hours and thereafter the supernatant was removed with a hand controlled vacuum

pump machine (Vacuum brand BVC 21 NT VARIO) to reduce the volume of the nematode

suspension to about 40ml for easy counting and quantification. The nematode population of both

M. chitwoodi and P. penetrans for each sample was obtained by counting all the nematodes

(eggs, juveniles and adults) in 40 ml of the supernatant.

As for the root subsample, the machine only extracts 500 ml out 1000 ml, therefore to obtain the

nematodes in whole subsample it was multiplied by a factor of 2. If the root system weight was

more than 5 g, the nematode final population (Pf) was extrapolated by multiplying the nematodes

in the subsample with actual root weight of the bulk sample divided by the subsample of the root.

For the soil subsample, the machine only extracts 100 cm3 out 200 cm3, therefore to obtain the

nematodes final population (Pf) in whole bulk sample it was multiplied by a factor of 20.

10

Figure 1: A schematic representation of automated zonal centrifuge machine (courtesy of

Wim Wesemael).

Data analysis

The number of egg masses of M. chitwoodi for screening tests and final nematode population

(Pf) for P. penetrans in both tests and M. chitwoodi in host status test were subjected to analysis

of variance (ANOVA) using a software program R x64 3.2.3. Differences among treatment

means were compared using Fisher’s least significant differences (LSD) at P < 0.05 and data was

normalized by log transformation. Nematode reproductive factor for each species was equally

calculated (Rf = Pf/Pi).

Based on the Rf, the plant cultivars were classified under five different categories (Schomaker et

al., 2013) as follows.

Non-host = (Rf <0.15), Poor host = (Rf < 1.0 ≥ 0.15), Maintenance host = (Rf ≤ 2.0 ≥1.0), Good

host (Rf≤ 4.0 ≥2.0) and Excellent host (Rf >4.0)

11

RESULTS

Resistance screening tests

Pratylenchus penetrans

Figure 2: Mean population of P. penetrans from a combination of organic and mineral soil

fractions of different green manure plant cultivars extracted 8 weeks after inoculation with 100

(juveniles and adults) in yellow tubes. Error bars show standard error and letters indicate

significantly similar and dissimilar groups (n= 6 and P ≤ 0.05). Pf = Nematode final population,

Pi = Nematode initial inoculation, BFT = Bird’s-foot trefoil.

A non-green manure and susceptible control (maize cv. LG 3220) supported the highest

nematode reproduction with the highest reproductive factor of 4.5 to P. penetrans in comparison

to other green manure plants and the difference was significantly different (P< 0.05) from all

other green manure plants (Fig. 2). Nematode reproduction rates on alfalfa cv. Alpha, bird’s-foot

trefoil cv. Bull and red clover cv. Lemmon were not significantly different with reproductive

factors of 2.83, 2.56 and 2.55 respectively. Bird’s-foot trefoil cv. Lotar and white clover cv.

Melital showed reproductive factors of 2.20 and 1.55 respectively and were not significantly

different from each other though significantly different from the other cultivars (Fig. 2). The

final populations of P. penetrans on bird’s-foot trefoil cv. Franco and arugula cv. Nemat were

b

cb

cd

b

cb

d

b

cd

d

a

100

0

100

200

300

400

500

600

BFT- Bull BFT-Lotar

BFT-Franco

Redclover

Whiteclover

Ryegrass Alfalfa Arugula Fodderradish

Maize

Mea

n n

emat

ode

fin

al p

op

ula

tio

ns

(Pf)

Pf

Pi

12

less than the initial population with reproductive factors of 0.93 and 0.89 respectively. Ryegrass

cv. Meltador and fodder radish line RsV79/80 showed the lowest reproductive factors of 0.3 and

0.24 respectively, with lowest final populations below the initial nematode population and

significantly different from the other crop hosts (Fig. 2).

Meloidogyne chitwoodi

Figure 3: Mean number of egg masses observed on roots of different green manure cultivars 8

weeks after inoculation with 100 second-stage juveniles of M. chitwoodi. Error bars show

standard error and letters indicate significantly similar and dissimilar groups (n= 10 and P ≤

0.05), BFT = Bird’s-foot trefoil.

A non-green manure and susceptible control tomato cv. Marmande was observed to have the

highest mean number of egg masses per root system (20.7) and was significantly different from

all other green manure plants (P< 0.05) (Fig. 3). Generally all the green manure plants did not

show high nematode multiplication with the observation of less number of egg masses on the

c

cc

b

c c c

c

c

a

0

5

10

15

20

25

30

Alfalfa Ryegrass Redclover

Whiteclover

BFT-Lotar

BFT-Bull BFT-Franco

Arugula Fodderradish

Tomato

Mea

n n

um

ber

of e

gg m

asse

s

13

root if not absent for some plant cultivars. Among the green manure plants White clover cv.

Melital had the highest mean number of egg masses (8.3). It was significantly different from the

rest of green manure plants and the susceptible control tomato cv. Marmande (P< 0.05).

Ryegrass cv. Meltador, arugula cv. Nemat, red clover cv. Lemmon and bird’s-foot trefoil cv.

Franco had mean number of egg masses greater than one but less than eight (3.1, 1.7, 1.6 and

1.6) respectively and they were not significantly different from each other. Alfalfa cv. Alpha,

bird’s-foot trefoil cv. (Bull and Lotar) and fodder radish line RsV79/80 had a mean number of

egg masses less than one and they were not significantly different from each other. Above all no

single egg mass was observed on the roots of fodder radish line RsV79/80 (Fig 3).

14

Host evaluation tests

P. penetrans

Table 3: The number of eggs, juveniles and adults in organic and mineral fraction and their

respective reproductive factor (Rf) on different green manure plant cultivars extracted 8 weeks

after inoculation with 500 P. penetrans (mixture of juvenile and adult stages). A susceptible

maize control and a fallow were subjected to the same inoculation.

Plants Root weight

(g)

Nematode population after 8 weeks Rf Host status

Organic

fraction

g-1 fresh

root

Mineral

proportion

2000 g-1 soil

Final

Populations

(Pf)

Arugula 3.65±0.27 6.0 236±116.10 de 258±110.97 e 0.52 Poor

Fodder radish 3.54±0.78 9.3 184±60.66 de 217.2±65.52 e 0.43 Poor

ESFR Mix 2.30±0.20 5.9 176±43.36 de 189.6±35.65 e 0.38 Poor

Ryegrass 14.23±1.44 7.2 256±63.88 de 358±81.895 e 0.72 Poor

Red clover 4.32±1.27 52.6 612±114.54 bc 839.2±145.74 bc 1.68 Maintenance

White clover 3.70±0.85 77.9 920±367.97 ab 1177.2±375.88 b 2.35 Good

Clover Mix 13.0±2.46 15.6 444±84.14 cd 647.2±167.47 cd 1.29 Maintenance

Fallow 76±38.47 e 76.0±38.47 e 0.15

Maize 29.47±3.66 57.47 940±167.33 a 2632.4±391.97 a 5.26 Excellent

Letters indicate significantly similar and dissimilar groups (n = 5 and P ≤ 0.05). Reproductive

factor (Rf) = final nematode population at harvest (Pf)/initial nematode inoculation (Pi). Values

are actual means with standard errors (±), ESFR = Arugula-fodder radish mix.

15

Figure 4: Mean population of P. penetrans (organic and mineral soil fraction) of different green

manure plant cultivars extracted 8 weeks after inoculation with 500 juveniles and adults. Error

bars show standard error and letters indicate significantly similar and dissimilar groups (n = 5

and P ≤ 0.05). Pf = Nematode final population, Pi = Nematode initial inoculation, ESFR =

Arugula-fodder radish mix.

Inoculation of the different green manure crops with P. penetrans resulted in varying final

populations (Fig. 4). Higher nematode numbers were recovered from the soil than in the roots

with the exception of susceptible maize cv. LG3220, where more nematode numbers were

recorded in the root fraction. Nematode populations did not correspond to root weights of green

manure cultivars. More nematode numbers were recovered in plant cultivars with a relative small

root system (low root weight), with the highest numbers recovered from roots of white clover

and red clover respectively (Table.3). Nematode reproduction was highest on susceptible control

maize (Rf 5.25) followed by white clover and red clover, (Rf 2.35 and 1.68) respectively.

Nematode population recovered from clover- English ryegrass mixture was slightly higher than

the initial population (Rf 1.29). However, nematode populations recovered from ryegrass cv.

ede e

ed

cb

b

cd

e

a

0

500

1000

1500

2000

2500

3000

3500

Arugula Fodderradish

ESFR Mix Ryegrass Red clover Whiteclover

Clover Mix Fallow Maize

Mea

n n

emat

ode

fin

al p

op

ula

tio

ns

(Pf)

OrganicproportionMineralproportionPi

16

Meltador, arugula cv. Nemat, radish line RsV79/80 and arugula- fodder radish (ESFR) mixture

were less than the initial populations (Rf 0.72, 0.52, 0.43 and 0.38 respectively). Least root

weight was observed on arugula- fodder radish (ESFFR) mixture and this corresponded with the

minimum number of nematodes recovered from the root fraction in comparison to other green

manure plants. Nematode populations were lowest on fallow treatment with a reproductive factor

of 0.15. There were significant differences in total nematode numbers recovered from the

different groups of green manure plant cultivars as indicated in Fig. 4 (letters indicating

significantly similar and dissimilar groups with P ≤ 0.05).

17

M. chitwoodi

Table 4: The mean number of eggs, juveniles and adults in organic and mineral fraction and their

respective reproductive factor (Rf) on different green manure plant cultivars extracted 8 weeks

after inoculation with 500 second-stage juveniles of M. chitwoodi. A negative control (fallow)

was subjected to the same inoculation.

Plant Root weight

(g)

Nematode population after 8 weeks Rf Host status

Organic

fraction

g-1 fresh

root

Mineral

proportion

2000 g-1 soil

Final

populations

(Pf)

Red clover 3.64±0.56 100.3 448±180.33 c 813.2±163.65 c 1.63 Maintenance

White clover 3.33±1.29 201.8 1312±263.29 a 1984±347.69 b 3.97 Good

Ryegrass 11.09±1.76 121.1 824±12.81 b 2167.6±396.91 b 4.33 Good

Clover mix 11.81±1.06 160.1 852±136.09 b 2743.2±332.22 a 5.49 Good

Fodder radish 3.02±0.22 0 236±38.47 cd 236±38.47 d 0.47 Non

Arugula 2.29±0.71 6.8 312±71.55 cd 327.6±64.92 d 0.65 Poor

ESFR mix 2.64±0.51 3.9 104±81.73 d 114.4±83.51 d 0.23 Poor

Fallow 124±26.08 d 124±26.07 d 0.25

Letters indicate significantly similar and dissimilar groups (n = 5 and P ≤ 0.05). Reproductive

factor (Rf) = final nematode population at harvest (Pf)/initial nematode inoculation (Pi). Values

are actual means with standard errors (±). Pf = Nematode final population, Pi = Nematode initial

inoculation, ESFR = Arugula-fodder radish mix.

18

Figure 5: Mean population of M. chitwoodi from a combination of organic and mineral soil

fraction of different green manure plant cultivars extracted 8 weeks after inoculation with 500

second-stage juveniles. Error bars show standard error and letters indicate significantly similar

and dissimilar groups (n = 5 and P ≤ 0.05). Pi = initial nematode inoculation, ESFR = Arugula-

fodder radish mix.

The clover-ryegrass mixture supported the highest nematode reproduction with the highest

reproductive factor of 5.49 and significantly different from all the green manure plants and a

fallow (P < 0.05). Ryegrass cv. Meltador and white clover cv. Melital equally had relatively high

reproductive factors of 4.33 and 3.97 respectively in comparison to other hosts and the recovered

c

b

b

a

dd

d d

0

500

1000

1500

2000

2500

3000

3500

Red clover Whiteclover

Ryegrass Clover Mix Fodderradish

Arugula ESFR Mix Fallow

Mea

n n

emat

ode

fin

al p

op

ula

tio

ns

(Pf)

Organicproportion

Mineralproportion

Pi

19

final populations were not significantly different from each other (P< 0.05) (Fig 5). Arugula cv.

Nemat, fodder radish line RsV79/80, arugula- fodder radish (ESFR) mixture and fallow did not

support nematode reproduction yielding final populations lower than the initial populations (Rf

0.65, 0.47, 0.23 and of 0.25) respectively The least number of nematodes was recovered from

arugula- fodder radish (ESFR) mixture though there were no significant differences with the

individual arugula cv. Nemat and fodder radish line RsV79/80 (Fig. 5).

DISCUSSION

Resistance screening of different green manure plants

This study presents results based on cultivar of a particular plant species. Alfalfa cv. Alpha had a

reproductive factor of 2.83. However, these findings are not in agreement with findings of a

greenhouse study carried out in Ontario, Canada which showed that the final population of P.

penetrans on alfalfa cv. Saranac was less than the initial population (Townshend & Potter, 1976).

This could be explained based on the fact that there could be differences in the level of resistance

between the two cultivars. Different Alfalfa cultivars are mostly like to influence nematode

invasion and multiplication differently.

The bird’s-foot trefoil cv. Bull, Lotar and Franco had reproductive factors of 2.56, 2.20 and 0.93

respectively. Therefore, bird’s-foot trefoil cv. Bull and Lotar are susceptible to P. penetrans

while bird’s-foot trefoil cv. Franco has a certain level of resistance to P. penetrans reproduction

similar to bird’s-foot trefoil cv. Empire which equally yielded a final population less than the

initial inoculation population in an experiment of Townshend and Potter (1976). Resistance to

nematode could be as a result of unknown plant proteins or unsuitable host cell for nematode

reproduction (Gheysen et al., 2006). This is the first report on screening of these three cultivars

for P. penetrans in temperate region. Therefore, pot greenhouse and field tests should be carried

out to assert the host status.

In many research studies, root gall index was used as factor for resistance screening of different

plants for the invasion of Melodoigyne species. Presence or absence of galls may not be

correlated with any Melodoigyne spp. reproduction (Al-Rehiayani & Hafez, 1998) as root galling

depends on physiological reaction of a particular plant (Wesemael, pers.comm). It is on this basis

the study decided to use egg mass numbers to assert invasion and reproduction of M. chitwoodi

on different green manure plant cultivars. However, present screening criterion of using number

20

of egg masses is backed up with root galling information which is available in many previous

studies.

Susceptible control and a non-green manure plant (tomato cv. Marmade) had the highest number

of egg masses from all the green manure plants and this is in agreement with the findings of

Kutywayo and Been (2006), where similarly a high number of egg masses and galls on the roots

were observed. Arugula cv. Nemat had a low mean number of egg masses compared to the

susceptible control tomato (cv. marmade). These findings substantiates the results of

Melakeberhan et al. (2006) who recorded more galling on tomato cv. Rutgers than arugula cv.

Nemat after inoculation with Meloidogyne hapla under greenhouse conditions. Few egg masses

were recovered from alfalafa cv. Alpha and this is in agreement with previous findings of

(Mermans, 2015) where fewer numbers of egg masses on alfalfa cv. Alpha under similar

greenhouse conditions were equally observed. Thus these two recent studies indicate that alfalfa

cv. Alpha has some level of resistance to M. chitwoodi multiplication. However, Griffin and

Rumbaugh (1996) reported that alfalfa cv. 1 and alfalfa cv. 2 had 60-80 % of the root tissues

galled hence contributing to reproductive factors of 12 and 10 respectively. This indicates that

not all alfalfa cultivars are resistant to the nematode and care should be taken when choosing a

cultivar for nematode management.

Among the bird’s-foot trefoil plants, high mean number of egg masses were observed on cv.

Franco and lowest on cv. Lotar. Griffin and Rumbaugh (1996) also reported the presence of galls

on bird’s-foot trefoil in greenhouse experiment with a gall index of 2.5 (60-80% roots galled)

and reproductive factor of 4.0, thus further highlighting its susceptibility to M. chitwoodi.

The mean number of egg masses for red clover cv. Lemmon were significantly different from

white clover cv. Melital, (1.6 and 8.3) respectively. This study contradicts with Griffin and

Rumbaugh (1996) who reported that red clover is susceptible and white clover resistant to M.

chitwoodi in the greenhouse. This could also be explained in terms of cultivar differences were

different cultivars influence nematode reproduction differently. No egg masses were found on

the roots of fodder radish line RsV79/80, deviating from the many galls observed in a

greenhouse test of fodder radish cultivars (Melodie, Adagio 5a-3 and Trez) by Ferris et al.

(1993). In a greenhouse study, Teklu et al. (2014) noted no differences between fodder radish

varieties (Radical, Doublet, Contra, Anaconda, Defender and Terranova) based on root galling

21

index. They concluded that all cultivars were partially resistant to M. chitwoodi. However,

fodder radish line RsV79/80 is a newly bred line which might have been developed with an

important trait of resistance to the important PPN of Europe. Egg masses were observed on the

root system of ryegrass cv. Meltador. Cook et al. (1999), showed that some cultivars of ryegrass

were heavily galled when inoculated with M. naasi. This could be of the reason that M. naasi has

been documented to be a host of many monocotyledonous plants (grasses and cereals)

(Wesemael et al., 2011)

For easy resistance screening of plants/crops or cultivars for Melodoigyne species, plant breeders

and Nematologists should use number of egg masses per root criterion. Egg masses are formed

on a susceptible plant while galling might not be present or difficult to observe. Secondly the use

of small yellow tubes in a greenhouse condition is simple, convenient and cost effective way of

screening.

Host evaluation different green manure plants

Maize cv. LG3220 (susceptible control) was found to be an excellent host and this is in

agreement with Kutywayo and Been (2006), who reported on maize cv. Husar as an excellent

host with reproductive factor of 6.4. During the pot experiment, maize exhibited a dense root

system which is of an advantage to the P. penetrans in accessing food and rapid multiplication.

Generally, the host evaluation study showed that ryegrass cv. Meltador is a poor host to P.

penetrans, findings which are in agreement with resistance screening based on the low

reproductive factor of 0.33. Abawi and Ludwig (1995) reported the similar results on treatment

of the nematode with ryegrass cv. Pennant.

Red clover cv. Lemmon and white clover cv. Melital are reported a maintenance and good host

respectively in the present study. This contradicts with the conclusions of Abawi and Ludwig

(1995) classifying both of them as intermediate hosts. However, their reproductive factor scaling

was different and maintenance and good hosts in this study are regarded as intermediate hosts in

their study. In a greenhouse study carried out in Canada, Papadopoulos et al. (2002) evaluated 18

cultivars and breeding lines of red clover and their reaction to inoculation of P. penetrans, only

one cultivar (AC Kingston) was highly susceptible to P. penetrans, cultivar Florex had low

levels of being invaded and three breeding lines (CRS 15,CRS 5 and CRS 11) registered both

low levels of invasion and low multiplication of the nematodes in the root. A four year field

22

micro-plot study in Abbotsford (British Columbia) found out that white clover was more

susceptible to P. penetrans (Vrain et al., 1996). Fodder radish line RsV79/80 is reported as a

poor host to P. penetrans in the current study, though at a species level, Hoek, (pers. comm)

indicates that fodder radish is regarded as a good host. Additionally, fodder radish cv. (Melodie

and Trez) were reported maintenance hosts to P. neglectus (Al-Rehiayani & Hafez, 1998). In

addition, planted Raphanus sativus (irrespective of cultivar) reduced population levels of P.

neglectus below 60% prior to planting potato in the field (Al-Rehiayani et al., 1999). Arugula cv.

Nemat is reported a poor host in this study. It is believed to be a trap crop hence suppressing

certain nematodes which enter the root with help of allelochemicals. The cultivar Nemat is

known to reduce plant-parasitic nematode populations and can be included in a crop rotation

scheme for organic farming (Curto et al., 2005; Kruger et al., 2013).

The clover- English ryegrass mixture is reported a maintenance host with a reproductive factor of

1.29, which is less than the reproductive factor of red clover cv. Lemmon (1.68) and white clover

cv. Melital (2.35) but greater than that of ryegrass cv. Meltador (0.72). Ryegrass cv. Meltador

was observed having a dense root system which covered the whole pot. It is therefore assumed to

have a contributing influence in reduction of nematode final populations of clover- English

ryegrass mixture. Ryegrass extensive root system and high forage is supplemented by the

Nitrogen supply from the leguminous clovers (Goh & Bruce, 2005) which in turn is of an

advantage to nematode reduction. Arugula- fodder radish mixture is a poor host along with the

singly planted arugula cv. Nemat and fodder radish line RsV79/80 to P. penetrans. Thus

integrating a combination of these green manure crops could be an advisable strategy in

nematode management nematode program.

It is important to note that green manure plant species well known for control of one type of

nematode may show susceptibility to other PPN (Cherr et al., 2006). Within a plant species,

different cultivars may not control a particular nematode species. Therefore, a green manure

mixture of different plant species is ideal in managing PPN and promoting organic farming.

The decline in nematode population in both fallows of M. chitwoodi and P. penetrans is in

agreement with the findings of (Kutywayo & Been, 2006; Wesemael & Moens, 2008), though

the experimental periods differ in weeks. Townshend (1984) revealed that nematodes while in

anhydrobiotic state can still thrive for about 110 weeks provided the loss of moisture is gradual.

Townshend (1984) suggested that temperature is a great abiotic factor in the persistence of the

23

nematode, as he reported that P. penetrans while in moist soils can survive at -4: 40-70˚C for 13

and 9 weeks respectively. Other factors for nematode persistence in a fallow include soil

moisture content, soil type and soil properties, the physiological age of the nematode and their

lipid reserves (Kutywayo & Been, 2006). The infectivity of the nematode decreases after a

prolonged state without food (Karssen et al., 2013), followed by dying of the nematode (Nježić

et al., 2014).

Egg masses and subsequent reproduction were noted within the root system of the green manure

crops which supported nematode invasion while absence of egg masses reproduction could have

been the result of nematode failure to invade and establish feeding sites in the root cells of some

manure crops. In this study red clover cv. Lemmon and white clover cv. Melital are classified

maintenance host and good hosts respectively. The plant species though not based on cultivars

were susceptible to M. chitwoodi with the reproductive factor of 9 and 10 respectively (Griffin &

Rumbaugh, 1996). Fodder radish line RsV79/80 is reported a non-host. This is in consensus with

findings of Ferris et al. (1993), where 9 of the 10 cultivars tested were poor or non-host to M.

chitwoodi with a reproductive factor less than 0.3. Presently, fodder radish line RsV79/80 being

a non-host complies with field micro-plot findings of (Al-Rehiayani & Hafez, 1998) for the two

fodder radish cultivars (Melodie and Trez) each with a reproductive factor of 0.1. Further this

study findings contradicts with the greenhouse evaluation for the three fodder radish cv.

(Melodie, Trez and Adagio 5a-3) as maintenance hosts with reproductive factors of 2.9, 2.8 and

1.7 respectively (Al-Rehiayani & Hafez, 1998). Recently, fodder radish varieties Anaconda,

Contra, Defender, Doublet and Terranova, known to have partial resistance, were evaluated and

their relative susceptibility were 0.17, 0.10, 0.42, 0.32 and 0.14% respectively (Teklu et al.,

2014). The M. chitwoodi populations reduced by more than 98% but (Teklu et al., 2014) could

not regard the varieties as non-hosts because their final nematode populations were dependent on

the initial nematode inoculation. Further field testing of RsV79/80 is thus recommended.

Ryegrass cv. Meltador is classified a good host in this current study with a reproductive factor

greater than 1.0. These findings are not in agreement with Hoek (pers. comm) that an English

ryegrass is a moderate host but whose crop damage is not clearly known. In a pot greenhouse

study Cook et al. (1999) confirmed a number of clones of ryegrass to be either resistant or

susceptible to M. naasi. Arugula cv. Nemat was identified as a poor host. This classification was

24

supported by the fewer number of nematodes in the root system and egg masses. The

significantly fewer nematode population densities of all stages extracted from arugula cv. Nemat

indicates that arugula hinders the development of all stages hence its capable of acting as trap

plant to manage M. hapla (Melakeberhan et al., 2006). Riga (2011) found a 99% reduction in M.

chitwoodi populations after planting potato as a follow-up crop in a greenhouse study. Riga

(2011) further narates that arugula on its own has not been able to control M. chitwoodi in the

field where lengthy season potatoes can support more than one generation of the nematode

species unlike in the green house. In South Africa, cv. Nemat, was reported a poor host and able

to inhibit M. javanica gall formation when applied as green manure in a glasshouse trial (Kruger

et al., 2015). The main allelochemical known for killing the nematodes is glucosinolates

particularly 4-methylthiobuthyl (Curto et al., 2005).

The current study presents the first report of clover- ryegrass mixture as a good host to M.

chitwoodi. It is reported that red clover cv. Lemmon, white clover cv. Melital and ryegrass cv.

Meltador are hosts to M. chitwoodi with reproductive factor of 1.63, 3.97 and 4.33 respectively.

This might have stimulated the high reproduction of the nematode in the mixture with a

reproductive factor of 5.49. It was observed that a good host ryegrass has dense root system

which covered the whole experimental pot, therefore its influence on the final nematode

reproduction of the mixture may be very fundamental than the two clovers. Obviously in the

field root development will be different and this might influence results. Therefore further field

testing is required. This initiative is in support of Peter and Rayns (2008) who recommended for

red clover-ryegrass, white clover/ryegrass or a complex containing several different cultivars of

several species of grasses and clovers. In promoting organic farming and management of M.

chitwoodi, this clover- English ryegrass mixture should not be used in the rotation. However,

clover- English ryegrass biofumigation investigation is recommended to further ascertain the

nematode levels especially in the field soils. Furthermore, the study presents the first report of

arugula- fodder radish mixture as a poor host with a reproductive factor less than that of the

individual plants, which seems logically true. The individual fodder radish and arugula are non-

host and poor host respectively, therefore the mixture is expected to reduce the nematode

reproduction. This would be the ideal green manure mixture to be used by farmers in

management of M. chitwoodi.

25

CONCLUSION AND RECOMMENDATION

Due to a policy to increase biodiversity farmers are financially rewarded if they use mixtures of green

manures. For farmers with P. penetrans and M. chitwoodi problems the options are limited and therefore

research on green manure mixtures is needed. Based on this study the classical clover-ryegrass mixtures

are not ideal but fodder radish (resistant cultivar) and arugula (cv. Nemat) can be successful.

Putting into consideration that different geographical areas have differing soil properties and

abiotic factors. It is essential first to select potential green manure plants that are adapted and

best fit into the local climatical crop rotation. It is recommended to carry out greenhouse or field

micro-plots for the various selected green manure plants to test for their host suitability to the

target pathogens before attempting larger scale field experience through farmers.

For a Belgian farmer willing to adopt organic farming, increase his or her crop yields and obtain

monetary incentives from the use of green manure mixtures provided by the European Union, it

should be a collaborative approach and advice from plant pathologists in different disciplines,

plant breeders and geneticists as well.

Acknowledgement

I do convey my appreciations to the Flemish government of Belgium (VLIR-UOS) for

sponsoring my master program. I take this moment to thank ILVO under the leadership of Prof

Nicole Viaene for the offer to work with the research team in the Nematology laboratories

especially Ms Nancy De Sutter. Many thanks to university of Ghent Nematology research unit

especially Prof wim Bert, Prof Decraemer, Ms Inge Dehennin and Emmanuelle De Bock for

their technical and administrative support during the whole period of my master program.

26

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