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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.
118 Ann Appl Biol 159 (2011)118129 2011 Rothamsted Research LtdAnnals of Applied Biology 2011 Association of Applied Biologists
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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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R.H. Manzanilla-L opez et al. Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites
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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Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites R.H. Manzanilla-L opez et al.
(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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R.H. Manzanilla-L opez et al. Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites
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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R.H. Manzanilla-L opez et al. Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites
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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R.H. Manzanilla-L opez et al. Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites
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
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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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