effects of crop plants on abundance of pochonia

7/29/2019 Effects of Crop Plants on Abundance of Pochonia

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Annals of Applied Biology ISSN 0003-4746

R E S E A R C H A R T I C L E

Effects of crop plants on abundance of Pochonia

chlamydosporia and other fungal parasites of root-knotand potato cyst nematodesR.H. Manzanilla-L opez1, I. Esteves2, S.J. Powers3 & B.R. Kerry1

1 Plant Pathology and Microbiology Department, Rothamsted Research, Harpenden, Hertfordshire, UK

2 Departamento of Life Sciences, Faculty of Sciences and Technology, IMAR-CMA, Coimbra, Portugal

3 Statistics Unit, Biomathematics and Bioinformatics Department, Rothamsted Research, Harpenden, Hertfordshire, UK

Keywords

Endophytes; Meloidogyne incognita;

Monographella cucumerina; oilseed rape;Paecilomyces lilacinus; rhizodeposits;

sugarbeet; wheat.

Correspondence

R.H. Manzanilla-L opez, Plant Pathology and

Microbiology Department, Rothamsted

Research, Harpenden, Herts AL5 2JQ, UK.

Email: [email protected]

Received: 2 November 2010; revised version

accepted: 5 April 2011.

doi:10.1111/j.1744-7348.2011.00479.x

Abstract

The effects of a host plant on reproduction/abundance of fungal populations

in relation to soil nutrients released by plants in the rhizosphere were studied.

Abundance in the soil and potato rhizosphere of the fungi Paecilomyces lilaci-

nus, Monographella cucumerina (CABI 380408) and Pochonia chlamydosporia var.

chlamydosporia (Pc280, potato cyst nematode biotype) and P. chlamydosporia var.

catenulata (Pc392, root-knot nematode biotype) were assessed. The different

ability of break crops (oilseed rape, sugarbeet and wheat) in the potato rotation

to support Pa. lilacinus, Pochonia isolates Pc280 and Pc392 and abundance of

the latter two isolates in soil and rhizosphere of potato plants infected with

Meloidogyne incognita were also studied. Potato chits and crop seedlings were

planted into boiling tubes containing 5000 chlamydospores or conidia g 1 in

acid washed sand (pH 6) and kept in a growth chamber at 20C, and 16 h of

light for up to 9 weeks. The abundance of the fungi in sand (fallow) differed

significantly between fungal species, being in general less abundant in theabsence than in the presence of the plant, although there was no interaction

between plant species and fungal isolate. There was evidence of a different

response to Me. incognita for Pc392 than for Pc280 but there was no significant

effect of the presence of the nematode on the rate of increase of the fungus.

Introduction

The fungus P. chlamydosporia (Goddard) Gams & Zare

(Clavicipitaceae) occurs saprophytically in soils and the

rhizosphere. It is has been reported as a parasite in eggs

of various invertebrates such as molluscs (Zare et al.,2001), helminths (Ara ujo et al., 2009a,b) and both ani-

mal and plant-parasitic nematodes (Braga et al., 2010;

Frassy et al., 2010). Pochonia is a potential biological con-

trol agent of plant endoparasitic nematodes of the genera

Meloidogyne spp. [root-knot nematodes (RKNs)], Nacob-

bus spp. (false RKNs) and Globodera and Heterodera spp.

(cyst nematodes). Globodera rostochiensis (Wollenweber)

and Globodera pallida Stone, commonly known as potato

cyst nematodes (PCN), are important pests in commer-

cial potato production in the UK (Atkins et al., 2003;

Tobin et al., 2008). Both PCN species multiply only on

solanaceous crops and weeds; hence, keeping soil free of

them for a number of years leads to a decline in nematode

populations (Whitehead & Turner, 1998). Integrated pest

management (IPM) for PCN includes the use of resistant

cultivars and nematicides in addition to crop rotation,although the latter is not always effective or econom-

ically viable. As a result, there is a need for effective

novel control strategies that can be included in an IPM

framework. The use of biological control agents such as

nematophagous fungi is a potential strategy to control

these pests (Kerry et al., 1993; Jacobs et al., 2003; Tobin

et al., 2008). However, the potential success of such a

biological control agent should be based on the careful

selection and combination of the fungal isolate biotype

(i.e. from the original nematode host) and host plant to

be included as break crops in the potato crop rotation.

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R.H. Manzanilla-L opez et al. Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites

Pochonia spp. are facultative parasites of nematode eggs.

Recent studies have shown that some Pochonia species

have an endophytic behaviour, which is a more inti-

mate relationship with the plant than just the saprophytic

behaviour so far attributed to the fungus in the rhi-

zosphere, and that this may be beneficial to the host

plants defence against other soil-borne pathogens (Bor-

dallo et al., 2002; Lopez-Llorca et al., 2002; Macia-Vicente

et al., 2009). It has been hypothesised that a change

or switch from the saprophytic to the parasitic phase

of the fungus may be related to nutrients released by

the plant into the rhizosphere. Plant root exudation,

or rhizodeposition, influences plant growth, resistance

to pests, beneficial symbioses, pathogen infection and

soil ecology in the rhizosphere via organic inputs and

depletion of large supplies of inorganic compounds. Rhi-

zodeposits are primarily composed of carbon-containingcompounds derived from photosynthetic products such as

small molecules (e.g. organic acids, amino acids, sugars),

secretions (enzymes), lysates, mucilage and quantities of

NO3 and NH4

+ (Bertin et al., 2003; Singh et al., 2004;

Wichern et al., 2008).

Plant species differ in their root exudates and rhi-

zodeposits, as well as in their ability to support

P. chlamydosporia growth in their rhizosphere and in

their susceptibility to infection by RKN (Kerry, 2000). A

potential bio-management strategy for nematode control

incorporates the use of P. chlamydosporia in combination

with selected cultivars of host plants (e.g. break crops),

which are less susceptible or resistant to the nematode

and that support extensive growth of the fungus in their

rhizosphere (Bourne et al., 1996; Bourne & Kerry, 1999).

The colonization of the root surface is closely linked to egg

mass production and changes in root exudation induced

by the nematodes (Bourne & Kerry, 1999). Hypothet-

ically, the fungi should translocate nutrients (including

carbon and nitrogen) across the mycelial network as far

as efficiency allows, and low numbers of nematode eggs

will maintain the fungi in the parasitic, rather than the

saprophytic, phase.

Pochonia chlamydosporia (= Verticillium chlamydosporium)

is one of the most important parasites responsible for thenatural control of both cereal and beet cyst nematodes

with precropping applications of the fungus surviving

long enough to kill nematode eggs and females that

develop on roots of spring-sown crops (Kerry et al., 1993).

Other PCN nematophagous fungi include Paecilomyces

lilacinus (Thom) Samson, 1974 and Monographella cuc-

umerina (Lindf.) Arx, 1984 (= Plectosphaerella cucumerina).

The impact of plant root exudates and rhizodeposition on

the parasitic activity of these two species is unknown.

Pa. lilacinus has been routinely isolated from infected

plant-parasitic nematode eggs and is one of the most

widely tested fungi for the control of root-knot and cyst

nematodes (Atkins et al., 2005). M. cucumerina has been

isolated from RKN and PCN nematodes (Atkins et al.,

2003) and the efficacy of the three fungi has been tested

for controlling PCN as part of an IPM regime by Jacobs

et al. (2003). Therefore, the objectives of the present study

were: (a) to assess if nutrients released in the potato

rhizosphere will increase abundance of the three PCN

nematophagous species: P. chlamydosporia, including iso-

lates of two Pochonia varieties, viz. P. chlamydosporia var.

chlamydosporia (Pc280, PCN biotype) and P. chlamydosporia

var. catenulata (Pc392, RKN biotype), Pa. lilacinus and

M. cucumerina (isolate CABI 380408), (b) to ascertain if

break crops in the potato rotation (oilseed rape, sug-

arbeet and wheat) differ in their ability to support

selected fungal isolates and if P. chlamydosporia occurs

as an endophyte within the roots of these crops; and(c) to assess if nematode infection by Me. incognita (Kofoid

& White, 1919) Chitwood, 1949 in potato plants pro-

vides P. chlamydosporia isolates with nutrients (different

from those obtained from the host plant alone) that may

enhance its reproduction and colonization of soil and

rhizosphere.

Materials and methods

Fungal isolates from nematodes

Pochonia chlamydosporia var. chlamydosporia isolate Pc280

(PCN biotype) and P. chlamydosporia var. catenulata isolatePc392 (RKN biotype) were obtained from the Rotham-

sted culture collection. The original host for isolate Pc280

(Jersey, UK) is a Globodera sp. and for the Cuban isolate

Pc392, a Meloidogyne sp.

Paecilomyces lilacinus (labelled as isolate PL LINK) was

used as a spore formulated wettable powder and prepared

according to the manufacturers instructions (Biological

Control Products SA, South Africa). The product, as

supplied by the manufacturer, had a concentration of

4 109 spore g1. M. cucumerina isolate CABI 380408

was obtained from CABI, UK. The original host for this

isolate was PCN from Jersey, UK (Atkins et al., 2003).

Production of inoculum (conidia and chlamydospores)

The different fungi and isolates were grown in selective

agar cultures as follows.

Paecilomyces lilacinus

A measure of 39 g of PDA (Oxoid, Basingstoke, UK),

10 g of sodium chloride and 28 mg pentachlornitroben-

dazole 99% (PCNB, Sigma-Aldrich, Milwaukee, MI,

USA) were added to 800 mL of distilled water and

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Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites R.H. Manzanilla-L opez et al.

autoclaved. Antibiotics included 50 mg of chlortetracy-

cline hydrochloride (Sigma-Aldrich), 100 mg of strepto-

mycin sulphate (Sigma-Aldrich) that were dissolved in

200 mL of tepid sterile distilled water (sdw) and 1 mL of

tergitol type NP-10 (Sigma-Aldrich) prior to being added

to the 800 mL of autoclaved agar.

Monographella cucumerina

A measure of 39 g of PDA (Oxoid), 10 g of sodium

chloride and 37.5 mg PCNB were added to 800 mL of

distilled water and autoclaved. Fifty microgram of chlorte-

tracycline hydrochloride (Sigma-Aldrich), 100 mg of

streptomycin sulphate (Sigma-Aldrich), 37.5 mg thiaben-

dazole (2-[4-thiazolyl]benzimidazole) (Sigma-Aldrich)

and 37.5 mg carbendazim 97% (Sigma-Aldrich) were

dissolved in 200 mL of tepid sdw and 1 mL of tergitol

type NP-10 before adding to 800 mL of autoclaved agar.

Pochonia chlamydosporia selective agar, potato dextrose

agar (PDA) and corn meal agar (CMA) were prepared

according to Kerry & Bourne (2002). PDA medium

was prepared for conidia production and CMA for

chlamydospores. Mass production of chlamydospores was

made using a rice culture.

Rice culture

Pochonia chlamydosporia isolates Pc280 and Pc392 were

cultured and incubated at 25C on rice substrate to pro-

duce chlamydospores (Kerry & Bourne, 2002; Hidalgo-

Daz, 2003). Twenty-five days after inoculation, the ricecontaining the chlamydospores was tipped from the flask

onto a sieve (250 m mesh pore) and rinsed with a jet of

water to collect the substrate and chlamydospores onto a

second sieve (10 m mesh pore). The sieve was blotted

underneath with a sponge and chlamydospores were col-

lected from the top surface of the mesh with a spatula.

Chlamydospores were then mixed with fine sand (low

iron; Fisher Scientific, Loughborough, UK) in a 10:1 w:w

ratio (sand:chlamydospores). One gram of inoculum was

added to 9 mL of water agar (0.05%) and thoroughly

mixed before chlamydospores were counted using a

haemocytometer (Marienfeld, Germany) and dilutionswere made to produce a final concentration of 5 103

chlamydospores mL1. Chlamydospore viability and ger-

mination percentage were evaluated on sorbose agar with

antibiotics (Esteves, 2007).

Meloidogyne incognita culture

Egg masses used in the experiments were taken from

tomato plants infested with Me. incognita. Nematode cul-

tures were started from a single egg mass and had been

kept in the glasshouse of Rothamsted Research for at least

5 years.

Inoculum preparation

A 20-m pore sieve was rinsed with 70% ethanol and

UV irradiated in a flow cabinet for 20 min. Conidia from

M. cucumerina and Pa. lilacinus were harvested separatelyfrom selective agar cultures grown in Petri dishes. Each

Petri dishwas flooded with 5 mL of sdw and the mycelium

was gently scraped using a sterile L-shaped glass rod. The

conidia suspension was poured onto the sieve mesh and

10 mL of sdw were added into the sieve; the conidia

suspension was then collected from a Petri dish placed

underneath. Conidia were counted under the microscope

using a haemocytometer and adjusted to a final spore

concentration of 5 103 mL1.

Experiment 1: fungal abundance (CFU) in potato

rhizosphere and acid washed sand

Quantification of P. chlamydosporia is difficult because of

the fact that the different life stages are neither com-

posed of approximately the same size units nor have the

same genetic contents (Mauchline et al., 2002). Although

quantitative PCR methods are increasingly used to quan-

tify the fungus in soil (Mauchline et al., 2002; Atkins &

Clark, 2004) and roots (Macia-Vicente et al., 2009), corre-

lation, for example, between colony-forming unit (CFU)

counts expressed as grams per dry weight to their equiv-

alent DNA quantities is still difficult because of various

factors including variable yields of DNA from samples

and amplification of DNA from fungal moribund material

that can give misleading results (Mauchline et al., 2002;

Manzanilla-L opez et al., 2009). The growth stage of the

fungus (e.g. DNA replication, hyphal growth, sporulation)

and root galling can also affect the number of gene copies

detected by PCR (Mauchline et al., 2002). Considering

that both methods can work up well to their theoreti-

cal limits in a sterile system (Mauchline et al., 2002), we

measured fungal abundance using the classic approach

of plate counting (CFU) that measures the abundance of

viable propagules of the fungus.

The abundance of two P. chlamydosporia isolates:

Pc280 (P. chlamydosporia var. chlamydosporia), Pc 392

(P. chlamydosporia var. catenulata), and single isolates ofPa. lilacinus and M. cucumerina was compared when the

carbon and nitrogen source for fungal growth was only

provided through the root system of potato plants. To

eliminate macro and micronutrients,20 kg of acid washed

coarse sand was saturated overnight with 1 M hydrochlo-

ric acid in a plastic container. Acid was washed away

and the coarse sand was thoroughly rinsed with dis-

tilled water until pH 6 was reached. To prepare each

experimental unit, 170 g of acid washed coarse sand

was weighed and placed in a reclosable polythene plastic

bag (180 200 mm) to which 5 mL of sdw was added



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to humidify the coarse sand before adding the chlamy-

dospores (5 103 g1 coarse sand) of P. chlamydosporia.

Coarse sand and chlamydospores were thoroughly mixed

in the bag and then transferred into each experimental

unit (20-cm long 4-cm diameter boiling tube). A similar

procedure was followed for Pa. lilacinus and M. cucumerina

except that inoculum for each fungus was added as 5 mL

of spore suspension (5 103 conidia g1 coarse sand).

Potato chits (cv. Cara) were planted in boiling tubes con-

taining the inoculated sand, wrapped in aluminium foil

and kept in a growth chamber for 4 weeks at 20C and 16

h of light (300 mol m2 s1) per 24 h. Controls consisted

of boiling tubes only containing fungus-inoculated coarse

sand that were sealed with Parafilm and watered regu-

larly to keep them moist (Fig. 1). Boiling tubes containing

the plants were watered daily. As soon as potato shoots

had emerged, they were manually sprayed twice a daywith foliar fertilizer (Phostrogen, pbi Home & Garden

Limited, Hertfordshire, UK) prepared according to the

manufacturers instructions, care being taken to avoid

leakage to the coarse sand. Four weeks later, the fresh

shoots and root system of each plant were measured and

weighed. Fungal populations from sand and roots (i.e.

rhizosphere) were isolated and 102 and 103 dilutions

were prepared and plated in selective agar to count CFU

(Kerry & Bourne, 2002) in triplicate from each boiling

tube. The CFU g1 coarse sand values were corrected to

the dry soil weight (Kerry & Bourne, 2002) according to

weight differences between dry and wet sand obtained

from 1 g of coarse sand taken per each experimental unit

(i.e. boiling tubes). The experiment was laid out as a ran-

domised block designwith four blocks. There wasa total of

10 treatments comprising a five by two factorial set: four

fungi and fallow (coarse sand) by two situations (plant

or no plant). There were four replicates per treatment (a

total of 40 experimental units). The analysis of CFU g 1

coarse sand and g1 root was made using ANOVA with a

square root transformation (to account for heterogeneity

of variance across the treatments) using GenStat Release

8.2 (VSN international Ltd, Hemel Hempstead, UK). A

stronger, natural log, transformation was used for the

CFU g1 roots. Following ANOVA, biologically relevantcomparisons of means were made using the least signifi-

cant difference (LSD) at the P = 0.05 level of significance.

Root and shoot variables were analysed similarly, but did

not require transformation.

Experiment 2: the ability of break crops to support

selected fungal isolates

On the basis of results obtained from Experiment 1,

P. chlamydosporia isolates Pc392 and Pc280 as well as

Pa. lilacinus were selected to be used in the second

Figure 1 Boiling tubesfilled with inoculated sand-grit,4-week-oldpotato

plants (left) and 4-week-old wheat plants (right).

experiment. Methods were similar to those described

in Experiment 1. Modifications included the use of acidwashed sand-grit mix (1:1 w/w) instead of coarse sand.

After the acid washed sand-grit had been inoculated

with each fungus, 1 g of sand-grit was taken at ran-

dom from 20 experimental units to assess initial CFU

counts (T1). Along with potato (Solanum tuberosum cv.

Maris Piper), for Experiment 2, spring cultivars of oilseed

rape (Brassica napus cv. Heros), sugarbeet (Beta vulgaris

cv. Dominica) and wheat (Triticum aestivum cv. Paragon)

were included as break crops. Seeds were surface sterilised

with commercial bleach (0.5%) and germinated at 25C

in Petri dishes containing nutritive agar [10 g L 1 glucose



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(Sigma-Aldrich), 0.1 g L1 yeast extract (Merck, Darm-

stadt, Germany), 0.1 g L1 peptone (Sigma-Aldrich) and

12 g L1 technical agar (Oxoid)] to ensure that they

were free from pathogens (Kerry et al., 1984). To syn-

chronise plant development, seeds of the different species

were germinated at 25C for different lengths of time to

provide seedlings with similar root length (3 cm long).

Seedlings were taken from Petri dishes and planted into

boiling tubes filled with sand-grit (170 g) previously inoc-

ulated with each fungus, and kept in a growth chamber as

described for Experiment 1. Four weeks later, plants were

removed from the boiling tubes and sand-grit was care-

fully removed from the roots. Shoots and root systems

were measured and weighed and CFU were counted

as in Experiment 1. The experiment was laid out as a

randomised block design with three blocks. Treatments

comprised the five crops (including fallow) with each ofthe three fungi and control. There were three replicates

per treatment, giving a total of 60 experimental units (i.e.

boiling tubes). Data for CFU g1 sand-grit (i.e. soil) and

CFU g1 root were recorded at the end of the experiment

(T2) and analysed, along with plant variables, as described

in Experiment 1.

Root staining

At the end of Experiment 2, sand-grit was removed

from roots and rinsed in sdw. Roots were cut into

1-cm-long segments. Roots per sample were wrapped

in an 11 11 cm piece of nylon voile, secured with

a wire and plunged into a beaker containing a boil-

ing solution of lactophenolethanol (1:2 v/v; Fisons and

Fisher Scientific, Loughborough, UK) for 10 15 min and

left overnight in a hooded cabinet at room temperature.

Afterwards, roots were transferred into another beaker

containing Trypan blue (BDH Stain, Poole, UK) lactophe-

nol (0.05%), stained for 45 min at 60C and left for

24 h in a hooded cabinet (Menendez et al., 1997). Sam-

ples were then rinsed in sdw and left in water-glycerin

(BDH, AnalaR; 1:1 v/v) within the hooded cabinet for

1 week to allow phenol evaporation. The nylon voile

was then removed and the stained roots were rinsed insdw, mounted in water-glycerin onto glass slides, covered

with a cover glass (22 50 mm) and sealed with nail pol-

ish. Slides were examined for root endophytes under the

microscope (Zeiss Axiophot, Carl Zeiss, Welwyn Garden

City, UK) at 20, 40 and 63 magnification.

Experiment 3: effect of nematode parasitism

On the basis of CFU counts from Experiments 1 and 2, for

isolates Pc280 and Pc392, a third experiment was carried

out to assess the effect of nematode parasitism on fungal

abundance. Potato chits (cv. Maris Piper) were planted

in acid washed sand-grit (pH 6) contained in Sterilin

(Sterilin Ltd, Aberbargoed, UK) skirted centrifuge tubes

(50 mL vol., blue lid) and placed in a growth chamber

for 1 week to allow roots to develop. Second-stage juve-

niles (J2) of Me. incognita were surface disinfected in 0.1%

Malachite green (Sigma-Aldrich) and 0.1% streptomycin

sulphate (Hooper, 1986). Each potato plant was inocu-

lated with 1000 J2 and returned to the growth chamber.

Ten days after J2 inoculation, plants were removed from

the Sterilin tubes and roots were rinsed carefully to wash

out those J2 that had not penetrated the roots. Plants

were transplanted into sand-grit inoculated with chlamy-

dospores as described before (Experiment 2) and returned

to the growth chamber for another 6 weeks. One gram of

sand was taken at random from 18 experimental units to

assess initial CFU counts (T1). The experimental designwas a randomised block with five blocks. There were

eight treatments comprising a three by two factorial set,

being the two fungi and a control (no fungus) each with

or without nematodes in the presence of potato, plus two

further control treatments for the fungi in the absence of

nematodes and potato. There were five replicates of each

treatment. Root and plant shoot lengths were recorded as

well as CFU g1 sand-grit and CFU g1 root. Number of

root galls and egg masses were also recorded. Egg masses

were hand-picked with fine forceps under a stereo micro-

scope and gently macerated in 2 mL of sdw contained

in a sterile glass homogeniser (Fisher Scientific). Eggswere then plated in Petri dishes containing 0.08% water

agar with antibiotics (Atkins et al., 2003; Esteves, 2007).

Plates were incubated for 3 days at 25 C and the per-

centage of infected eggs was assessed. The percentage

of eggs parasitised (P%) by the fungus was logit trans-

formed, including an adjustment to account for zero

recordings [log10 ((P% + 1)/(101 P%)], for ANOVA.

Other variables recorded were analysed as for the pre-

vious experiments. Pearson correlations were calculated

between the different variables.

Results

Experiment 1

In this experiment, the proliferation of the different

fungi in the presence or absence of a potato plant is

considered. CFU data obtained from coarse sand and rhi-

zosphere in order to assess the effect of the crop plant on

the abundance of the fungal species revealed significant

(P< 0.001, F-test) main effects and interaction between

absence/presence of a plant and the fungal species. The

fungi reacted differently to the addition of a potato plant,

M. cucumerina and P. chlamydosporia var. catenulata (Pc392)



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Table 1 Experiment 1: means of CFU g1 acid washed sand and CFU g1 roots from potato rhizosphere for Monographella cucumerina, Paecilomyces

lilacinus, Pochonia chlamydosporia var. chlamydosporia (Pc280) and P. chlamydosporia var. catenulata (Pc392)a

Fungi

TreatmentFallow(control) M. cucumerina Pa. lilacinus

P. chlamydosporia var.chlamydosporia (Pc280)

P. chlamydosporia var.catenulata (Pc392)

No. potato (sand only) 0.0 0.0 193.4 67.5 103.2

Potato and sand 0.0 15.0 185.2 67.9 202.8

Potato rhizosphere 0.0 3.2 13.5 14.5 14.4

aMean values are square root of CFU g 1 of acid washed sand LSD (P = 0.05) = 35.53, SED = 17.32, df = 27, n = 4 and CFU log (CFU roots +1) g 1

roots from potato rhizosphere LSD (P = 0.05) = 5.17, SED = 2.28, df = 9, n = 12.

being most different (Table 1). With a potato plant, Pa.

lilacinus, Pc280 and Pc392 CFU coarse sand counts were

higher and significantly different (P< 0.05, LSD) from

M. cucumerina but Pa. lilacinus and Pc392 were not sig-

nificantly (P> 0.05, LSD) different amongst themselves(Table 1). There was a significant difference (P< 0.05,

LSD) between treatments (potato versus no potato) only

for isolate Pc392. Although CFU counts increased for

Pc392 in coarse sand in the presence of a potato plant,

CFU counts remained at similar levels for Pc280 regard-

less of presence/absence (Table 1). For the natural log

of CFU counts for roots there was a significant differ-

ence (P = 0.003, F-test) between isolates: Pa. lilacinus,

Pc280 and Pc392 were significantly different (P< 0.05,

LSD) from M. cucumerina with higher number of CFU

countsbut were not significantlydifferent (P> 0.05, LSD)

amongst themselves. There were no statistical differences

(P> 0.05, F-test) between treatments for plants shoot

and root variables (data not shown).

Experiment 2

In this experiment, the effect of different break crops

on the proliferation of the fungi is considered. First, in

order to resolve if Pc280 levels remained much the same

because of survival of chlamydospores rather than minor

increments of the isolate, CFU counts for both isolates

were assessed at planting (T1) and at the end (T2) of

the second experiment. At planting (T1) there was a

significant difference (P< 0.001, F-test) in square rootCFU g1 of acid washed sand-grit between isolates, Pc280

being significantly different from the other fungi (P 0.05, LSD). Significant

differences occurred between fungi (P< 0.001, F-test)

for final square root CFU g1 acid washed sand-grit at

T2, but there was no significant difference due to crops

(P = 0.988, F-test) or due to an interaction between crops

and fungus (P = 0.180, F-test). For the square root of

CFU counts for roots there was a significant interaction

between crops and fungi (P = 0.003, F-test) with a strong

main effect of fungus (P< 0.001, F-test) but not of crops(P = 0.460, F-test). Investigating this interaction, there

was a strong effect of Pa. lilacinus for oilseed rape and

sugarbeet and this fungus gave the highest CFU value in

the potato rhizosphere. The isolate Pc392 was not assessed

on sugarbeet because plants died before the experiment

was completed, but this isolate produced the largest CFU

counts in the wheat rhizosphere (Table 2).

Table 2 Experiment2:meansofCFUg 1 acidwashedsand-gritandg1 rootof Pochoniachlamydosporia var. chlamydosporia(Pc280), P. chlamydosporia

var. catenulata (Pc392) and Paecilomyces lilacinus from break crops at initial assessment (planting, T1) and at final assessment (4 weeks after planting,

T2)a

T1 (n) Fallow (T2) Oilseed rape ( T2) Potato ( T2) Sugarbeet (T2) Wheat (T2)Mean forisolates (T2)

Fungus Sand-grit Sand-grit Root Sand-grit Root Sa nd-gri t Root Sa nd-gri t Root Sand-grit Root Sand-grit

Control 0.0 (18) 0.0 0.0 0.0 0.0 6.1b 0.0 0.0 0 0.0 0.0 1.2

Pa. lilacinus 30.2(15) 382.7 0.0 410.9 2152 441.2 669.0 386.4 1916 416.5 957 407.5

Pc280 84.2 (15) 152.7 0.0 179.5 372 155.7 570.0 162.5 261 165.3 292 163.1

Pc392 39.5 (9) 488.7 0.0 435.1 574 420.2 573.0 438.4 ND 380.2 1488 432.5

aMeans are square root values of CFU g1 acid washed sand-grit and g1 root. T1: CFU sand-grit: LSD (Pa. lilacinus versus Pc280, P = 0.05) = 16.74,

SED = 7.84, df = 15; LSD (Pa. lilacinus versus Pc392 or Pc280 versus Pc392, P = 0.05) = 19.33, df = 15. T2: CFU sand-grit LSD (only for comparison of

means for isolates, in bold, P = 0.05) = 32.30, SED = 15.95, df = 38, n = 45; CFU roots (for all comparisons) LSD ( P = 0.05) = 897.70, df = 38, n = 9;

ND = not determined because of plants having died.bPossible contamination.



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Table 3 Experiment 2: means of root length (cm) for combinations of isolates of Pochonia chlamydosporia var. chlamydosporia (Pc280),

P. chlamydosporia var. catenulata (Pc392) and Paecilomyces lilacinus with crops

Oilseed rape Potato Sugarbeet Wheat Means for isolates

Rootlength

Shootlength

Rootlength

Shootlength

Rootlength

Shootlength

Rootlength

Shootlength

Rootlength

Shootlength

Control (no fungus) 22.5 4.2a 30.5 7.7 4.8 2.1 43.2 14.7 25.2b 7.2

Pa. lilacinus 20.7 4.6 26.8 7.9 15.8 3.2 31.2 17.2 23.5 8.2

Pc280 25.8 8.3 26.3 6.9 21.2 3.7 56.2 13.7 32.4 8.2

Pc392 21.6 4.6 28.2 7.2 17.4 2.5 40.0 14.7 26.8 7.2

Means for crops 22.6b 5.4 27.8 7.4 14.8 2.9 42.6 15.1

aShoot length: LSD (comparisons between two-way table of means, P = 0.05) = 2.98; SED = 1.45, df= 25, n = 3.bRoot length: LSD (comparison of means for crops or for isolates only, in bold, P = 0.05) = 6.53;SED = 3.17, df= 26, n = 12.

There was a main effect of crop (P< 0.001, F-test) and

fungi (P = 0.030, F-test) on root length (Table 3). Isolates

Pc280 and Pc392 were associated with the longest roots,but root length for Pc280 and Pa. lilacinus was not signif-

icantly different from the control (P> 0.05, LSD). Only

root length for isolate Pc280 was significantly different

from the control (P< 0.05, LSD). Overall, there was little

real effect on root length due to the presence of fungus.

Following a marginally significant main effect of fungus

(P = 0.029, F-test) for root weight, there were, however,

no significant differences (P> 0.05, LSD) in root weight

(data not shown) when comparing fungi to control.

Finally, for shoot length, a marginally significant inter-

action was found to occur between fungal isolates and

crops (P

= 0.036,F

-test). In particular, isolate Pc280 was

associated with greater shoot length for oilseed rape, and

Pa. lilacinus was associated with greater shoot length for

wheat, than the other two fungi and the control (Table 3).

Fungus endophytic behaviour

Chlamydospores were observed on the surface of the

roots of all crops. However, microscopical observations

of the endophytic root behaviour of the two isolates of

Pochonia were made only for potato and wheat (a total

of 12 root samples plus controls because of the poor

growth of plants from the other crops). Hyphae of the

fungus were found on the rhizoplane of both crops, oftenassociated with chlamydospores as well as intercellular

hyphae (Fig. 2), and forming steps along cell walls, as

reported for barley (Hordeum vulgare) by different authors

(Bordallo et al., 2002; Lopez-Llorca et al., 2002; Mont-

fort et al., 2005). Conidia and conidiophores were also

observed in epidermal cells.

Experiment 3

Here the effect of crop nematode parasitism on fungal

abundance is investigated. The ANOVA of the square

root of sand-grit CFU at T1, showed a statistically

marginal effect of fungus (P = 0.056, F-test), with a

higherinoculum level for isolate Pc280 (82.5) on the inoc-ulated sand-grit in comparison with isolate Pc392 (36.1)

and control treatments (0.0) [LSD (P = 0.05) = 62.94,

SED = 19.78, df = 3, n = 6]. Final sand-grit CFU counts

(T2) showed that, despite the difference in CFU at T1,

isolate Pc392 reached greater CFU numbers than isolate

Pc280 (Table 4). ANOVA of the square root of sand-grit

CFU at T2, partitioning the various sources of varia-

tion, showed a significant difference between the two

isolates in the absence of potato (P< 0.001, F-test), a

significant effect of fungus overall (P< 0.001, F-test), a

weak effect of the presence of nematodes (P = 0.089,

F-test), and a weak difference between the two fungi

(P = 0.073, F-test). Most importantly, there was a sig-

nificant interaction between fungus and presence of Me.

incognita (P = 0.005, F-test) having accounted for the

control treatments without potato, so the presence of the

nematode affected the two isolates in different ways. For

Pc280 no difference was found in presence/absence of

nematodes (132.6 vs 144.5). For Pc392 a difference was

found (198.6 vs 128.7; Table 4).

On roots, the presence of the nematode was associated

with lower square root CFU g1 for both isolates, with

mean values of 406 for Pc392 and 212.5 for Pc280, in

comparison with 467.6 (Pc392) and 272.9 (Pc280) in

the absence of the nematode [LSD (P = 0.05) = 75.52,SED = 36.20, df = 20]. A similar result was obtained for

Pc280 CFU in sand-grit, but a contrary result was obtained

for Pc392, which had higher CFU in sand-grit in the

presence than in the absence of the nematode (Table 4).

Longer roots and shoots were produced by potato plants

in the presence of the nematode (Table 5). On average,

the highest number of galls (64.2 17.87, n = 5) and

egg masses (29 10.23, n = 5) per root occurred in

control plants (i.e. without the fungus) followed by Pc392

(60 16.11 galls and 28.6 7.25 egg masses, n = 5) and

Pc280 (57.4 23.45 galls and 28.4 12.97 egg masses,



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A B

C D

Figure 2 Micrographs of Pochonia chlamydosporia on wheat roots. (A) control without fungus, (B) Pc392 mycelium inside root cells, (C) Pc280

chlamydospores and (D) Pc392 hyphae.

Table 4 Experiment 3: means of square root CFU (sand-grit) for

combinations of Pochonia chlamydosporia var. chlamydosporia (Pc280)

and P. chlamydosporia var. catenulata (Pc392) and presence (+) and

absence () of Meloidogyne incognita

Isolate Plant Nematode CFU

Pc280 166.2a

Pc280 + 144.5

Pc280 + + 132.6

Pc392 398.8

Pc392 + 128.7

Pc392 + + 198.6

Control (no fungus) + 0.0

aFor comparison of means: LSD (P = 0.05) = 38.99, SED = 19.04,

df= 28, n = 15.

n = 5). The number of eggs produced in each egg mass

ranged between 0 and 60 (data not shown).

Using only data from plants in the presence of the

nematode for the two fungal isolates, there were signif-

icant (P< 0.05, F-test) negative correlations (r, Pearson,

Table 5 Experiment 3: means of root and shoot length (cm) in

presence of Meloidogyne incognita and Pochonia chlamydosporia var.

chlamydosporia (Pc280) and P. chlamydosporia var. catenulata (Pc392)

combinations

Me. incognita

(+) root

Me. incognita

() root

Me. incognita

(+) shoot

Me. incognita

() shoot

Pc280 29.5 20.9 8.7 7.4

Pc392 26.0 25.0 8.1 7.6

Control (no

fungus)

31.2 26.1 7.9 6.0

Means 28.9a 24.0 8.2b 7.0

aRoot length comparing Me. incognita (+ versus ) means in bold

over fungi (P = 0.019, F-test, LSD (P = 0.05) = 3.96, SED = 1.89, df= 9,

n = 15).bShoot length comparing Me. incognita (+ versus ) means in bold

over fungi (P = 0.063, F-test, LSD (P = 0.05) = 1.3, SED = 0.62, df= 19,

n = 15).

n = 15 pairs) of the sand-grit CFU with root weight, root

length and shoot weight. Therefore, when the nematode

was present, as CFU values in the sand-grit went up, the



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plant measures went down. Egg masses were only signif-

icantly correlated (r = 0.524, P = 0.045, n = 15, F-test)

with root length. In the absence of nematodes the corre-

lations of CFU with plant measures were not significant

(P> 0.05, F-tests). For data where egg masses and fungal

infection were present, a positive correlation (r = 0.791,

P = 0.020, n = 8, F-test) was found between percentage

of egg infection and numbers of CFU in the root. The

correlation of egg infection with CFU in the sand-grit was

not significant for the two isolates (r = 0.582, P = 0.130,

n = 8, F-test) but also indicative of a positive relationship

between fungus and nematode. However, care should be

taken as these results are only based on eight pairs of

values.

CFUs incremented over time (T1 to T2) of the experi-

ment (6 weeks) but there was no significant effect of the

presence of the nematode on the rate of increase of Pocho-nia (P = 0.558, F-test). However, across both Pochonia

isolates, the presence of the nematode gave a higher rate

of CFU acquisition (52 vs 31 square root CFU week1, s =

39.62, df = 4), and isolate Pc392 had a higher, although

not significantly different (P = 0.209, F-test) rate of

increase in CFU over time in comparison with Pc280

(76.3 vs 47.6 square root CFU week1, s = 18.0, df = 3).

Discussion

Plants and their rhizodeposits sensu lato are an important

source of C and N for soil microbiota to maintain some

entomopathogenic and nematophagous fungi, in a sapro-

phytic stage in soil (Bruck, 2010). In the present study, we

have used a simple and economical approach to assess, in

the absence of other source of N, C and other nutrients,

except for the plant (and, later on, the nematode), the

effect of different host plants on the abundance of three

different species of nematophagous fungi in the sand and

rhizosphere. The approach developed worked better for

potato and wheat plants, which produced larger foliage

surfaces earlier in their development and throughout the

duration of the experiments, than sugarbeet or oilseed

rape. Foliar feeding alone was not enough to sustain fur-

ther development of the young plants of oilseed rape andsugarbeet.

CFU data from Experiment 1 showed that the plant

had an important effect in increasing abundance of the

different fungal species and isolates in coarse sand and

roots in comparison with fallow (i.e. no plant). Of the

three fungal species tested, Pa. lilacinus was the most

abundant and M. cucumerina was the least abundant in

coarse sand. This result supports a previous report for

the latter species as a poor competitor in an assay to

control PCN that included Pa. lilacinus and P. chlamy-

dosporia (Jacobs et al., 2003). Comparison of isolates of

the two varieties of P. chlamydosporia, revealed that iso-

late Pc280 (P. chlamydosporia var. chlamydosporia) was less

abundant than isolate Pc392 (P. chlamydosporia var. catenu-

lata) despite evidence of higher Pc280 CFU initial counts

related to chlamydospore germination (data not shown).

CFU counts of Pc280 at the beginning and the end of

the experiment remained almost at the same level (or

had a negligible increment). Hence it was not affected by

presence of the plant, and remained viable in the absence

of plants, as has been also reported by Mauchline et al.

(2002). The poor saprophytic behaviour shown by Pc280

in comparison with Pc392, agrees with Mauchline et al.

(2004) who also found that Pc280 was present in simi-

lar numbers at the start and end of experiments, when

applied to healthy and PCN-infested tomato plants. How-

ever, differences between colonization of soil, rhizosphere

and eggs parasitism can vary between Pochonia isolates.According to Siddiqui et al. (2009), although Pc280 was

a less effective soil and rhizosphere colonizer, it was the

most virulent isolate on RKN and PCN eggs.

There was no significant difference in sand-grit CFU

counts for Pa. lilacinus and Pc392 due to break crop

species as shown by Experiment 2 but an interaction was

found to occur between fungal isolates and crops. Of the

crops tested, potato (Solanaceae) has been reported as a

good host for the fungus, with up to 14 125 CFU g 1

soil on sandy loam with potato (Bourne et al., 2004),

whereas wheat (Gramineae) has been reported as a poor

host (Kerry, 2000). However, the status of wheat as a

poor host of Pochonia will need to be revised in view of

the high CFU counts obtained on roots for Pc392 as they

were more abundant in the rhizosphere of wheat, rather

than that of potato. Cereals such as wheat release C and

N in good quantities in their rhizodeposits and these may

range between 4.3% and 56% of total plant N (Wichern

et al., 2008). Such crops are more likely than other to

support the fungus in higher CFU numbers in soil and

rhizosphere but wheat and its residues can also influence

weeds, pests, diseases and other soil microbes because

of the allelochemical compounds produced (Bertin et al.,

2003; Bais et al., 2006).

Of the four break crops tested, there is scantinformation available on the effect of sugarbeet and

oilseed rape on P. chlamydosporia isolates and abundance

of other nematophagous fungi. However, one study, on

the influence of green manuring on egg pathogens of

Heterodera schachtii with three intercrops in crop rotation

with sugarbeet, showed that the antagonistic potential of

the egg pathogenic fungi was much greater in a rotation

(sugarbeetwheat) than in a sugarbeet monoculture

(Pyrowolakis et al., 1999). Our data showed that there was

a strong effect of oilseed rape (Brassicaceae) and sugarbeet

(Chenopodiaceae) on Pa. lilacinus abundance. Reports on



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the effect of brassicas on the fungus have shown both

positive and negative growth results on P. chlamydosporia

isolates, situations that ultimately affect both the

nematode and fungus (Bourne et al., 1996, 2004).

Biotypes of P. chlamydosporia from cyst- and root-

knot nematodes can have important differences in their

biology, host preference (at the plant and nematode

level), physiology and ecology requirements (Mauchline

et al., 2004; Siddiqui et al., 2009). Some isolates of

P. chlamydosporia grow rapidly while others grow poorly

in different soils following application (Kerry et al., 1993;

Siddiqui et al., 2009). Our results showed that RKN bio-

type Pc392 had a higher growth rate than PCN biotype

Pc280 and that this, under the experimental conditions

used, may be linked to host preference as Meloidogyne spp.

is the preferred host for RKN biotype Pc392 and PC280 is

a poor parasite of RKN (Mauchline et al., 2002). Accord-ing to Siddiqui et al. (2009) marked biotype differences

in abundance in soil and CFU numbers can be generally

greater in nematode-infested soils than in non-infested

soils. Our results showed the opposite effect with lower

CFU counts in soil in the presence of the nematode, thus

giving support to the hypothesis that low levels of nema-

tode eggs will maintain the fungi in the parasitic, rather

than the saprophytic, phase.

There was little correlation between CFU and the

potato plant measurement data, but our results also

showed that when the fungus and nematodes occurred

together there was a negative effect on plant growth vari-

ables (i.e. root and shoot length), in contrast to results

obtained from Experiment 2 where plants had longer

roots and shoots in the presence of the fungi but with-

out nematodes. The negative correlation of the sand-grit

CFU counts of the fungus and the plant growth found

in the presence of the nematode could be explained by

the fungus proliferating more in the roots than in the

sand-grit when there were nematode eggs in the vicinity

of the rhizosphere for it to infect; however, it may also be

related to the fitness cost of saprophytic versus parasitic

growth and virulence (Siddiqui et al., 2009). Different

isolates from biotypes of RKN and PCN (such as Pc280)

of P. chlamydosporia var. chlamydosporia can increase thefresh weights of the shoots and roots of potato plants to

differing degrees (Siddiqui et al., 2009), a phenomenon

also observed in Experiment 2. However, nematode infes-

tation, although perhaps not affecting shoot weight, may

reduce mean root biomass (Siddiqui et al., 2009).

Colonisation of the root surface by the fungus is closely

linked to egg mass production and is thought to be related

to changes in root exudation and systemic effects on the

plant because of nematode infection of the root system

(Bourne & Kerry, 1999; Yeates, 1999). Mauchline et al.

(2004) pointed out that differential growth of Pochonia

isolates indicated both the great variation in the ability of

P. chlamydosporia isolates to use root exudates saprophyt-

ically, and the qualitative and/or quantitative difference

in nutrients available in the rhizosphere of plants. Abun-

dance may be related to C and N provided alone by the

plant in rhizodeposits that are used by the fungus to

support its saprophytic behaviour. In the present study,

fungi were more abundant in the plant rhizosphere than

in soil and, under our experimental conditions, another

source of nutrients (macro/micronutrients) could have

been provided through root leakage/exudates induced

by the nematode whose feeding sites (i.e. giant cells)

act as a metabolic sink for nutrients withdrawn from

the plant (Bais et al., 2006). Attraction by the fungus

to a richer source of energy (e.g. carbohydrates) such

as plant rhizodeposits, nematode gelatinous matrix (i.e.

glycoproteins) and nitrogen from eggs may support thehypothesis that nutrition (use of C and N) is one of the

factors involved in switching from saprophytic to parasitic

behaviour. The presence of Me. incognita was associated

with higher CFU mean values for isolate Pc392 in the soil

in comparison with isolate Pc280, but CFU differences

may also be due to differential saprophytic and parasitic

abilities of the two isolates of P. chlamydosporia varieties.

Two of the fungal species included in our study have

been reported as endophytes. Pochonia is a facultative par-

asite that can also behave as an endophyte with species

such as P. rubescens increasing root length of barley (H.

vulgare) seedlings and reducing Gaeumannomyces graminis

var. triticiroot colonization (Montfort et al., 2005; Lopez-

Llorca et al., 2008). Pa. lilacinus endophytic behaviour

has been reported elsewhere (Rumbos & Kiewnik, 2006)

but there is no information available in the literature

regarding the endophytic potential of P. chlamydosporia

isolates for break crops such as oilseed rape, potato,

sugarbeet and wheat. In the present study, preliminary

results using light microscopy showed P. chlamydosporia

var. chlamydosporia (Pc280) and P. chlamydosporia var.

catenulata (Pc392) to be a root endophyte in the wheat

and potato roots. This observation deserves further inves-

tigation using different approaches, including molecular

(Schulz & Boyle, 2006). Potential endophytic root colo-nization by egg-parasitic fungi such as Pochonia may open

an opportunity to infect eggs of plant endoparasitic nema-

todes inside the roots and also to explore new application

methods of the fungus to the plant (i.e. seed) and to the

soil (Lopez-Llorca et al., 2008).

Bio-management strategies for control of nematodes

that incorporate the use of P. chlamydosporia in com-

bination with selected cultivars of host plants that are

less susceptible or resistant to the nematode and support

extensive growth of the fungus in their rhizosphere, can

be improved by taking into consideration not only the



11/12


inclusion of a poor host for the nematode, but also a

host that releases rhizodeposits that could support fungal

growth in the rhizosphere and an endophytic behaviour.

This bio-management strategy can be combined with

other IPM practices and biological control agents incor-

porated as part of an IPM for PCN. According to Jacobs

et al. (2003), some potato growers already apply two

control measures for PCN, a fumigant in the autumn fol-

lowed by a granular nematicide in the spring (at a cost

of approximately 900 ha1) and so separate applica-

tions of two biological control agents, such as Pa. lilacinus

and Pochonia spp., may be feasible (Jacobs et al., 2003).

Tobin et al. (2006) have also shown the potential of using

P. chlamydosporia to control PCN in potato crops grown

under commercial field conditions.

Acknowledgements

Rothamsted Research is an institute of the Biotechnology

and Biological Science Research Council of the UK. This

project was funded by DEFRA Link Project LK0966.

The authors thank Dr Penny R. Hirsch and Mr Ian

Clark for the technical advice and the Bioimaging and

Visual Communications Unit (Rothamsted Research) for

preparing the figures.

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