Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella

mycological research 113 (2009) 1312–1321

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Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella Ariel W. GUZMA´N-FRANCOa,c,*, Suzanne J. CLARKb, Peter G. ALDERSONc,1, Judith K. PELLa a

Department of Plant and Invertebrate Ecology, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Department of Biomathematics and Bioinformatics, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK c School of Biosciences, University of Nottingham, Loughborough, Leicestershire LE12 5RD, UK b

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abstract

Article history:

The entomopathogenic fungi Zoophthora radicans and Pandora blunckii co-occur in field

Received 19 December 2008

populations of Plutella xylostella and, therefore, are likely to interact during the infection

Received in revised form

process. We have investigated the possible outcomes of these interactions in the labora-

20 August 2009

tory. Using four isolates, two of each fungal species, inter-specific interaction experiments

Accepted 28 August 2009

were done in Petri dishes and on intact plants. In Petri dish experiments, larvae were inoc-

Available online 16 September 2009

ulated directly using sporulating mats of mycelium, both species had the same opportunity

Corresponding Editor:

to infect and only the relative concentration of conidia of each pathogen species applied

Richard A. Humber

was manipulated. In the intact plant experiments, larvae were placed onto fungus-contaminated plants, inoculation was passive and the probability of infection by either or both

Keywords:

species of fungi depended on larval activity and proximity to inoculum. In the Petri dish

Entomopathogenic fungi

experiment, the species with the largest concentration of conidia out-competed the other

Entomophthorales

regardless of virulence, and results were similar in the intact plant experiment. The ecolog-

Inter-specific interactions

ical implications for competition or co-existence of these two pathogens in the field are discussed. ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Zoophthora radicans (Brefeld) Batko and Pandora blunckii (Bose & Mehta) Humber are entomopathogenic fungi from the order Entomophthorales (Entomophthoromycotina). Their life cycles begin with conidia produced on conidiophores that emerge from host cadavers. These primary conidia are discharged actively. If they land on a host, they can germinate

and infect the host by direct penetration through the cuticle. Once inside the host the fungus proliferates as protoplasts and/or hyphal bodies that ultimately invade all tissues and kill the host (Pell et al. 2001). Some entomophthoralean fungi, e.g., Z. radicans, also produce resting spores that are thickwalled and remain within the body of the dead host (e.g., Glare et al. 1989; Yeo et al. 2001). This attribute is highly variable even amongst isolates of the same species (Glare et al. 1987). Resting

* Correspondence to: Ariel W. Guzma´n-Franco, Postgrado en Fitosanidad, Campus Montecillo, Colegio de Postgraduados, Km 36.5 Carretera Me´xico-Texcoco, Montecillo, Texcoco, Estado de Me´xico 56230, Me´xico. Tel.: þ52 5959520200x1609; fax: þ52 5959520267. E-mail address: [email protected] 1 Present address: The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia. Tel.: þ603 8924 8212. 0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2009.08.016

Competition and co-existence of Zoophthora radicans and Pandora blunckii

spores can persist for many years and are thought to be produced for survival during unfavourable abiotic conditions, e.g., during winter (Pell et al. 2001). The entomophthoralean fungi Z. radicans, P. blunckii and their host, the diamondback moth Plutella xylostella L, co-occur in space and time (Riethmacher & Kranz 1994), suggesting that it is extremely likely these two fungal species will interact in P. xylostella populations. Interactions between two or more pathogen species or genotypes attacking the same host are now considered to be common, or even the rule (Read & Taylor 2001). These interactions have the potential to modify the population dynamics of a particular insect–pathogen relationships (Thomas et al. 2003). Such interactions can result in exclusion of one of the pathogens or co-existence and the outcome may be determined by differences in virulence. If the virulence of both pathogens is similar, both may co-infect via scramble competition (e.g., Nowak & May 1994). The outcomes from a dual-inoculation and infection could also be greatly influenced by abiotic factors such as temperature (e.g., Inglis et al. 1997; Fargues & Bon 2004) and biotic factors inherent to each pathogen, such as variation in developmental rate, concentration ratio, host range (e.g., Koppenho¨fer & Kaya 1996; Perlman & Jaenike 2001) and spatial distribution (Massey et al. 2004). In a previous study (Guzma´n-Franco 2005), in vitro interactions amongst different isolates of Z. radicans and P. blunckii were studied. The outcomes demonstrated that P. blunckii interfered with growth of Z. radicans. In particular, P. blunckii isolate NW449 had a significant negative effect on the growth of all Z. radicans isolates evaluated. This isolate, along with another isolate of P. blunckii and two Z. radicans isolates, were selected to study the outcomes when dual-inoculated onto P. xylostella larvae at different spatial scales. Quantifying the relationship between Z. radicans and P. blunckii in P. xylostella is ecologically and practically important. It contributes to our understanding of the parameters that modulate competition between natural enemies and underpins the development of entomopathogens for microbial control programmes against P. xylostella.

Material and methods

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a reference isolate to previous studies (e.g., Pell et al. 1993; Furlong et al. 1995). All isolates were grown on SEMA medium (Sabouraud dextrose agar supplemented with egg yolk and milk; Wilding & Brobyn 1980). The cultures were incubated at 20  C in darkness for 15 d before use. Prior to experimentation, cultures were subcultured no more than three times after retrieval from liquid nitrogen.

In vivo inter-specific interactions between Zoophthora radicans and Pandora blunckii isolates against P. xylostella larvae in Petri dishes Inoculation was done directly onto larvae by exposing them beneath sporulating fungal plugs, thereby ensuring that each larva received conidia. Dual-inoculations of both species were made in all possible isolate combinations. Due to the difficulty in delivering a pre-determined concentration for each combination of isolates, different times of exposure were used in order to obtain different proportions of conidia of each species in nine different treatments (Table 1). Exposing insects to actively sporulating plugs for different periods of time to achieve different inoculation doses is a standard technique for evaluating entomophthoralean fungi (e.g., Pell et al. 1993; Shah et al. 2003, 2004). Actively sporulating plugs were prepared by placing 9 mm diameter plugs of each isolate (cut using a cork borer) in the lids of 90 mm diameter Petri dishes containing damp filter paper on the base and then incubating these at high humidity and 20  C in darkness for 18 h prior to experimentation. After this time they were actively sporulating. Batches of 20 early third instar larvae of P. xylostella were each inoculated with the nine different treatments (Table 1) by placing them in 30 mm Petri dish bases each containing a cabbage leaf disk embedded abaxial side uppermost in 1.5 % water–agar (2 ml) and exposing them to actively sporulating plugs either from Z. radicans or P. blunckii or both (see below). The larvae fed on the leaf during inoculation and did not come into direct contact with inoculum. A 10 mm diameter glass coverslip was placed in the centre of each cabbage leaf during inoculation to capture conidia. After inoculation, the coverslips were removed, fixed with 10 % cotton blue in lactophenol on glass slides and the concentration and identity (in dual inoculations) of conidia estimated by counting conidia

Plutella xylostella culture The P. xylostella colony was reared in Perspex cages (0.5 m2  50 cm) at 23  C (16L:8D) in the quarantine area of the Rothamsted Research insectary. The colony was maintained on Chinese cabbage plants. The original colony came from the Philippines in the 1980s and has been maintained at Rothamsted Research continuously since then.

Fungal isolates Zoophthora radicans isolates NW250 and NW386, and Pandora blunckii isolates NW449 and NW454, all from the Rothamsted Research collection, were used in these experiments. All isolates, except NW250, were isolated from Plutella xylostella on brassicas in Guanajuato State, Mexico. NW250 was isolated from P. xylostella on brassicas in Malaysia, and was included as

Table 1 – Exposure times (min) used to create nine different treatments representing different relative proportions of conidia of each species present. Zr [ Zoophthora radicans, Pb [ Pandora blunckii. Treatment

Zr

Pb

1 2 3 4 5 6 7 8 9

45 5 45 5 45 5 0 0 0

5 45 45 5 0 0 45 5 0

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from ten fields of view with a 1 mm2 eyepiece graticule using the 10 microscope objective. The control treatment (Treatment 9, Table 1) was maintained under the same conditions as the other treatments for 90 min but without inoculum. Dual-inoculations were carried out in 20 cm diameter plastic Petri dishes. Two 30 mm diameter Petri dish lids were attached in the lid of the large Petri dish (20 cm) using double sided adhesive tape. One of the 30 mm diameter Petri dish lid contained seven Z. radicans sporulating plugs and the other lid the same quantity of P. blunckii sporulating plugs. Two 30 mm diameter Petri dish bases, containing a cabbage leaf disk as described above were placed in the base of the large 20 cm diameter Petri dish beneath the lids containing inoculum. The 20 cm Petri dish contained 50 ml of 1.5 % water–agar to maintain high humidity throughout. During dual-inoculation, the base of the 20 cm diameter Petri dish was kept stationary and only the lid, containing the two 30 mm diameter lids with the fungal plugs, was rotated. With this rotation the same batch of 20 larvae was inoculated with both species of fungi sequentially; the different proportions of conidia from each fungal species were obtained by exposing the larvae for different lengths of time. After inoculation, each batch of larvae was transferred to ventilate transparent polystyrene boxes (124  82  22 mm) (Stewart Group Holdings Ltd., Surrey, England) containing fresh Chinese cabbage leaf material. The polystyrene boxes were incubated at high humidity for the first 24 h to ensure germination of the conidia. All treatments were incubated at 20  C in a 12:12 L:D regime. Mortality was recorded daily for six days. During scoring, all larvae were allocated to one of the following categories: living, dead due to Z. radicans, dead due to P. blunckii, dead due to a combination of both pathogens (dual-infected) and dead due to unknown mortality factors. In order to identify the cause of death of the larvae, each larva was individually placed in the base of a 30 mm diameter Petri dish containing 2 ml of 1.5 % water agar. These Petri dishes were inverted and incubated at 20  C for 24 h in a 12:12 L:D regime. The inverted position allowed the collection of conidia on the lid of the Petri dish. After the 24 h incubation period, all lids were fixed with 10 % cotton blue lactophenol and covered with a coverslip. The presence or absence of conidia and identity of one or both pathogens assessed microscopically using conidia morphology. No conidial quantification was made. Simultaneously, the presence or absence of resting spores inside all cadavers was identified microscopically. The nine treatments for each isolates combination were carried out on the same day and the whole experiment was repeated on three separate occasions.

Statistical analysis Each isolate combination was analysed separately. In each combination, the data were analysed using logistic regression (assumed to follow a binomial distribution) with each cause of death as a response variable–either mortality by Pandora blunckii, Zoophthora radicans, both pathogens or unknown cause of mortality (UCM)–and a sample size equal to the number of individuals tested. Each number of deaths was a proportion of the total number of insects tested. The different concentrations obtained for each isolate in each combination were allocated into three categories,

A. W. Guzma´n-Franco et al.

‘‘Control’’ when no fungus was inoculated, ‘‘Low concentration’’ with all the concentrations obtained with the least time of inoculation (five minutes), and ‘‘High concentration’’ with all the concentrations obtained with longest inoculation time (45 min). When analysing mortality due to infection by either fungus alone or in combination (dual-inoculated), only data from the treatments where specific fungi were inoculated were included. Hence, the total number of data points and degrees of freedom for treatment terms and the residual differed depending on the variable analysed. Where necessary, the presence of more variability in the data than expected under the binomial assumptions (over-dispersion) was allowed for by testing the ratio of the treatment mean deviance to the residual mean deviance against the F-distribution, rather than testing the usual treatment deviance against the c2 distribution. All the statistical analyses were carried out using the statistical package GenStat (Genstat Committee 2000).

In vivo inter-specific interactions between Zoophthora radicans and Pandora blunckii isolates against Plutella xylostella larvae on Chinese cabbage plants The objective of this experiment was to quantify the interactions that may occur between the two pathogens in P. xylostella populations in which the larvae were not directly inoculated by the fungi but received inoculum passively, if they passed close to an actively sporulating inoculum source. Only three isolates were used, the Z. radicans isolates NW250 and NW386, and the P. blunckii isolate NW449. Two-week-old Chinese cabbage plants were placed individually in unventilated cylindrical cages made from a transparent flexible plastic sheet over an 11  8 inch seed tray with an absorbent mat in the base to allow watering of the plant. The cages were not ventilated to avoid cross-contamination of fungal conidia amongst treatments. Watering was minimized to avoid excess condensation inside the cages. Fifteen early third instar larvae of P. xylostella were first placed on each plant. The larvae were allowed a period of two hours to establish feeding sites and then, according to treatment, different combinations of fungi were placed on the plants. The treatments were as follows NW250 þ NW449, NW386 þ NW449, NW250, NW386, NW449 and control with no fungus. In the treatments containing two fungal species, two 9 mm plugs cut from 15 d old growing colonies of each species were placed separately on two of the largest leaves (two plugs of one species on one leaf and two plugs of the other species on a second leaf). In the single species treatments two plugs of the species under evaluation were placed together on only one leaf. The control was without either fungus. The cages were incubated at 20  C and a 12:12 L:D regime. Mortality was recorded on day four. All the cadavers were placed individually in 30 mm diameter Petri dishes on 2 ml of 1.5 % water agar at 20  C and 12:12 L:D regime and infection (presence of discharged conidia or resting spores) determined as described previously. All the remaining living larvae from each treatment were maintained in ventilated transparent polystyrene boxes (124  82  22 mm) until day five to record any further mortality. The experiment was run according to

Competition and co-existence of Zoophthora radicans and Pandora blunckii

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a completely randomised block design with six treatments and three blocks. The experiment was repeated on two occasions.

Statistical analysis The analysis was the same as for the Petri dish experiment, where all the isolate combinations were analysed together. Each cause of death was analysed separately. All the statistical analyses were carried out using the statistical package GenStat (Genstat Committee 2000).

Results In vivo inter-specific interactions between Zoophthora radicans and Pandora blunckii isolates on Plutella xylostella larvae in Petri dishes In all isolate combinations, different concentrations of conidia were successfully achieved using different times of exposure to sporulating plugs. Conidial concentrations from the longest exposure time (average of 126.78 conidia/mm2) were consistently larger than the concentrations obtained from the shorter exposure times (average of 16.03 conidia/mm2).

Dual inoculation with Zoophthora radicans isolate NW250 and Pandora blunckii isolate NW449 For P. blunckii isolate NW449 (Fig 1A) and Z. radicans isolate NW250 (Fig 1B) the largest proportion of larvae becoming infected occurred when the highest inoculation concentration was applied (F1,10 ¼ 4.73, P ¼ 0.055; F1,10 ¼ 5.45, P ¼ 0.042 for P. blunckii and Z. radicans respectively). This result was regardless of the presence of conidia of the other species, either for P. blunckii (F2,10 ¼ 0.45, P ¼ 0.648) or Z. radicans (F2,10 ¼ 1.47, P ¼ 0.277). For P. blunckii the largest proportion of infected larvae occurred when no conidia of Z. radicans were present, and this proportion was reduced in the presence of Z. radicans conidia (F2,10 ¼ 4.74, P ¼ 0.036) (Fig 1A). For Z. radicans, the largest proportion of infected larvae occurred when no conidia of P. blunckii were present, and this proportion was reduced in the presence of P. blunckii conidia (F2,10 ¼ 23.90, P < 0.001) (Fig 1B). The proportion of dual-infected larvae was small, less than 0.01, which was equivalent to 1 %, in all treatments. The proportion did not vary with concentration of P. blunckii (F1,6 ¼ 2.52, P ¼ 0.164) or Z. radicans (F1,6 ¼ 0.08, P ¼ 0.790) conidia. The proportion of cadavers with UCM varied amongst the concentrations of P. blunckii conidia (c22 ¼ 5.75, P ¼ 0.003). The presence of P. blunckii (either at low or high conidia concentration) increased the number of cadavers with UCM compared to the control treatment where no conidia were applied (Fig 1C). The proportion of cadavers with UCM did not vary amongst the different concentrations of Z. radicans (c22 ¼ 0.52, P ¼ 0.592).

Dual inoculation with Zoophthora radicans isolate NW250 and Pandora blunckii isolate NW454 For P. blunckii (Fig 2A) and Z. radicans (Fig 2B) the largest proportion of larvae becoming infected occurred when the highest inoculation concentration was applied (F1,10 ¼ 9.97, P ¼ 0.010;

Fig 1 – Proportion of Plutella xylostella larvae infected. (A) Infection caused by isolate NW449 (Pandora blunckii) alone when Low and High concentrations of NW449 were applied in combination with three concentrations (Low, High and No conidia) of isolate NW250 (Zoophthora radicans). (B) Infection caused by NW250 (Z. radicans) alone when Low and High concentrations of NW250 were applied in combination with three concentrations (Low, High and No conidia) of NW449 (P. blunckii). (C) UCM in all nine treatments. Error bars represent 95 % confidence intervals back-transformed from the logistic scale. Minimal dual-infection (<1 %) occurred for any treatment. Number in brackets represents the average conidial concentration (conidia/mm2).

F1,10 ¼ 5.85, P ¼ 0.036 for P. blunckii and Z. radicans respectively). This result was regardless of the presence of the conidia of the other species, either for P. blunckii (F2,10 ¼ 0.28, P ¼ 0.761) or Z. radicans (F2,10 ¼ 0.30, P ¼ 0.747). For P. blunckii the largest proportion of infected larvae occurred when no conidia of Z. radicans were present, and this proportion was reduced in the presence of Z. radicans conidia (F2,10 ¼ 5.93, P ¼ 0.020) (Fig 2A). For Z. radicans the largest proportion of infected larvae occurred when no conidia of P. blunckii were present, and this proportion was reduced in the presence of P. blunckii conidia (F2,10 ¼ 17.19, P < 0.001) (Fig 2B). Whilst the overall proportion of dual-infected larvae was similar for the high and low concentrations of P. blunckii

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A. W. Guzma´n-Franco et al.

Fig 2 – Proportion of Plutella xylostella larvae infected. (A) Infection caused by isolate NW454 (Pandora blunckii) alone when Low and High concentrations of NW454 were applied in combination with three concentrations (Low, High and No conidia) of isolate NW250 (Zoophthora radicans). (B) Infection caused by NW250 alone when Low and High concentrations of NW250 were applied in combination with three concentrations (Low, High and No conidia) of NW454 (P. blunckii). (C) Dual-infection by NW250 (Z. radicans) and NW454 (P. blunckii). No dual-infection occurred for High NW454 and Low NW250. (D) UCM in all nine treatments. Error bars represent 95 % confidence intervals back-transformed from the logistic scale. Number in brackets represents the average conidial concentration (conidia/mm2).

conidia (c21 ¼ 1.82, P ¼ 0.178), the overall proportion was greater for the high concentrations of Z. radicans conidia than for the low concentrations (c21 ¼ 18.49, P < 0.001) (Fig 2C). The proportion of cadavers with UCM did not vary amongst any concentration of either P. blunckii (F2,16 ¼ 2.43, P ¼ 0.120) or Z. radicans conidia (F2,16 ¼ 1.37, P ¼ 0.282). The proportion of cadavers with UCM was the same in the no fungus control and the treatments where different concentrations of conidia were applied (Fig 2D). The average proportion of cadavers with UCM was 0.133 which is equivalent to 13.3 %.

P. blunckii conidia (c21 ¼ 1.38, P ¼ 0.239), the overall proportion of larvae infected was greater for the high concentration of Z. radicans conidia than for the low concentration (c21 ¼ 4.83, P ¼ 0.028) (Fig 3C). The proportion of cadavers with UCM did not vary amongst any concentration of Z. radicans conidia (F2,16 ¼ 0.79, P ¼ 0.548). The largest proportion of cadavers with UCM were obtained in the treatments where different concentrations of P. blunckii conidia were applied compared to the control treatment where no conidia were applied (F2,16 ¼ 7.54, P ¼ 0.005) (Fig 3D).

Dual inoculation with Zoophthora radicans isolate NW386 and Pandora blunckii isolate NW449

Dual inoculation with Zoophthora radicans isolate NW386 and Pandora blunckii isolate NW454

For P. blunckii isolate NW449 (Fig 3A) and Z. radicans isolate NW386 (Fig 3B) the largest proportion of larvae becoming infected occurred when the highest inoculation concentration was applied (c21 ¼ 42.40, P < 0.001; c21 ¼ 18.20, P < 0.001 for P. blunckii and Z. radicans, respectively). However, for both isolates, these results varied in the presence of conidia (at different concentrations) of the other species (P. blunckii (c22 ¼ 32.91, P < 0.001) and Z. radicans (c22 ¼ 2.83, P ¼ 0.059)). For P. blunckii the largest proportion of infected larvae occurred when no conidia of Z. radicans were present, and this proportion was reduced in the presence of Z. radicans conidia (c22 ¼ 32.91, P < 0.001) (Fig 3A). For Z. radicans the largest proportion of infected larvae occurred when no conidia of P. blunckii were present, and this proportion was reduced in the presence of P. blunckii conidia (c22 ¼ 34.33, P < 0.001). Whilst the overall proportion of dual-infected larvae did not vary between the high and low concentrations of

For P. blunckii isolate NW454 (Fig 4A) and Z. radicans isolate NW386 (Fig 4B) the largest proportion of larvae becoming infected occurred when the highest inoculation concentration was applied (F1,10 ¼ 4.79, P ¼ 0.054; c21 ¼ 14.73, P < 0.001 for P. blunckii and Z. radicans respectively). For P. blunckii this result was regardless of the presence of conidia (at different concentrations) of Z. radicans (F2,10 ¼ 2.85, P ¼ 0.105), but it varied for Z. radicans, where the proportion was affected by the presence of conidia (at different concentrations) of P. blunckii (c22 ¼ 5.15, P ¼ 0.006). For P. blunckii the largest proportion of infected larvae occurred when no conidia of Z. radicans were present, and this proportion was reduced in the presence of Z. radicans (F2,10 ¼ 11.24, P ¼ 0.003) (Fig 4A). For Z. radicans, the largest proportion of infected larvae occurred when no conidia of P. blunckii were present, and this proportion was reduced in the presence on P. blunckii conidia (c22 ¼ 34.39, P < 0.001).

Competition and co-existence of Zoophthora radicans and Pandora blunckii

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Fig 3 – Proportion of Plutella xylostella larvae infected. (A) Infection caused by isolate NW449 (Pandora blunckii) alone when Low and High concentrations of NW449 were applied in combination with three concentrations (Low, High and No conidia) of isolate NW386 (Zoophthora radicans). (B) Infection caused by isolate NW386 (Z. radicans) alone when Low and High concentrations of NW386 were applied in combination with three concentrations (Low, High and No conidia) of NW449 (P. blunckii). (C) Dual-infection by isolates NW386 (Z. radicans) and NW449 (P. blunckii). (D) UCM in all nine treatments. Error bars represent 95 % confidence intervals back-transformed from the logistic scale. Number in brackets represents the average conidial concentration (conidia/mm2).

Fig 4 – Proportion of Plutella xylostella larvae infected. (A) Infection caused by isolate NW454 (Pandora blunckii) alone when Low and High concentrations of NW454 were applied in combination with three concentrations (Low, High and No conidia) of isolate NW386 (Zoophthora radicans). (B) Infection caused by isolate NW386 (Z. radicans) alone when Low and High concentrations of NW386 were applied in combination with three concentrations (Low, High and No conidia) of NW454 (P. blunckii). (C) Dual-infection by isolate NW386 (Z. radicans) and NW454 (P. blunckii). (D) UCM in all nine treatments. Error bars represent 95 % confidence intervals back-transformed from the logistic scale. Number in brackets represents the average conidial concentration (conidia/mm2).

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The overall proportion of dual-infected larvae was similar for the high and low concentrations of P. blunckii (c21 ¼ 2.45, P ¼ 0.117) (Fig 4C); however, the overall proportion was greater for the high concentrations of Z. radicans conidia than for the low concentrations (c21 ¼ 8.58, P ¼ 0.003). The proportion of cadavers with UCM did not vary amongst any concentration of Z. radicans conidia (c22 ¼ 1.86, P ¼ 0.156). The presence of the P. blunckii (either at low or high concentrations) increased the number of cadavers with UCM compared to the control treatment (Fig 4D) (c22 ¼ 5.78, P ¼ 0.003).

In vivo inter-specific interactions between Zoophthora radicans and Pandora blunckii isolates on Plutella xylostella larvae on Chinese cabbage plants Proportion of Plutella xylostella larvae infected only with Pandora blunckii isolate NW449 The proportion of larvae infected with P. blunckii isolate NW449 alone was greater on plants inoculated with this isolate alone compared to plants where P. blunckii was dual-inoculated with either Z. radicans isolate NW250 or NW386 (Fig 5A), (F1,10 ¼ 6.14, P ¼ 0.033). On the dual-inoculated plants, the proportion of larvae infected with P. blunckii was the same regardless of whether it had been dual-inoculated with Z. radicans isolate NW250 or NW386 (F1,10 ¼ 1.59, P ¼ 0.236).

Proportion of Plutella xylostella larvae infected only with Zoophthora radicans isolates NW250 or NW386 The average proportion of larvae infected with Z. radicans isolate NW250 on single or dual-inoculated (with Pandora blunckii isolate NW449) plants were the same as the average proportion of larvae becoming infected with Z. radicans isolate NW386 when it was single or dual inoculated with P. blunckii isolate NW449 (F1,15 ¼ 3.45, P ¼ 0.083). The greatest proportion of larvae infected with either Z. radicans isolates always occurred when no P. blunckii conidia were present (F1,15 ¼ 11.88, P ¼ 0.004) (Fig 5B) and was the same for both Z. radicans isolates (F1,15 ¼ 0.14, P ¼ 0.712).

Proportion of Plutella xylostella larvae dual-infected with Zoophthora radicans isolates NW250 or NW386 and Pandora blunckii isolate NW449 The proportion of dual-infected larvae did not vary between plants inoculated in the NW250/NW449 or NW386/NW449 combinations (F1,5 ¼ 1.83, P ¼ 0.234). The overall proportion of dual-infected larvae was generally very small, with 0.02 and 0.009 for the NW250-NW449 and NW386-NW449 combinations respectively.

Proportion of cadavers with unknown causes of mortality (UCM) In all the treatments where P. blunckii was inoculated (either single or dual-inoculated with either of the Z. radicans isolates), the proportion of cadavers with UCM was always greater than in the absence of P. blunckii (F1,25 ¼ 18.18, P < 0.001) (Fig 5C), and this was independent of which Z. radicans isolate was present (F2,25 ¼ 0.12, P ¼ 0.887). The proportion of cadavers with UCM did not vary amongst the treatments with Z. radicans (F2,25 ¼ 0.43, P ¼ 0.656).

Fig 5 – (A) Proportion of larvae infected with NW449 (Pandora blunckii) alone obtained from larvae on plants inoculated only with NW449 and the average proportion obtained from plants inoculated with NW449 and NW250 or NW386. (B) Proportion of larval population infected with Zoophthora radicans on plants inoculated either with NW250 or NW386 only (Z. radicans) and plants dual-inoculated with NW449. (C) UCM in all nine treatments. Error bars represent 95 % confidence intervals back-transformed from the logistic scale.

Discussion During simultaneous dual-inoculation in Petri dishes, the probability of either of the two species infecting a group of Plutella xylostella larvae was negatively affected by the presence of the other species. In a previous study, all four isolates were virulent against P. xylostella (Guzma´n-Franco 2005). Although statistical differences in virulence were found between and within species, these differences were insufficient to affect the outcomes of the interactions. Furthermore, the ability of the Pandora blunckii isolate NW449 to interfere with the in vitro growth of Zoophthora radicans isolates found previously (Guzma´n-Franco 2005)

Competition and co-existence of Zoophthora radicans and Pandora blunckii

was not found in the in vivo studies described here. However, conidial concentration did have a strong effect on the outcomes: the larger the concentration of conidia, the stronger the effect. In direct dual-inoculation, both isolates had the same opportunity to infect the host without consideration of differences in transmission abilities. When Z. radicans isolate NW250 was dual-inoculated with either P. blunckii isolate, the overall outcome of the intra-host competition was that larvae appeared to be infected only with one isolate. This suggests that one isolate excluded the other completely. Although it is possible that unidentifiable mycelia of the other species were present within the host they were ultimately unable to produce conidia and could, therefore, be considered to be effectively excluded because the numbers of transmission stages (or conidia) are the measure of pathogen fitness and transmission capability (Ebert & Weisser 1997; Solter et al. 2002). The similarity in virulence of these isolates (NW250 and NW449/454) (Guzma´n-Franco 2005) suggests that the results were more concentration-ratio related, which means that the one with the larger number of conidia out-competed the other one regardless of the virulence of either isolate. This may be because larger concentrations of conidia infecting a host will produce a larger pool of mycelium more quickly (Hughes et al. 2004), leading to competition by exploitation, where the more abundant pathogen will use the resources faster and deprive the other isolate of them (Rayner & Weber 1984). Some dual-infected larvae were observed, although the proportions were always low, normally below 0.2 (equivalent 20 %). Dual infection is likely to have occurred as a result of a dual inoculation with a similar concentration of conidia from both fungal species that then shared the host equally. A host can be an environment with substantial spatial and temporal heterogeneity in resource availability, mortality factors for the pathogen and indigenous microflora (Smith & Holt 1996). Massey et al. (2004) found that when two species of virulent entomopathogenic bacteria–Photorhabdus asymbiotica (Fisher-Le Saux et al. 1999) and Xenorhabdus nematophilus (Poinar & Thomas)–were inoculated simultaneously on Galleria mellonella larvae, they did not exclude each other. This was as a result of the spatial structure within the larvae, allowing different species to dominate different patches thereby avoiding competitive exclusion. The dual-infected P. xylostella larvae showed very limited external fungal growth and sporulation on the host body, if any. Sometimes sporulation only occurred in small patches and did not cover the whole larva, personal observation, and very few conidia were produced. This external sporulation in patches may suggest an effect of the internal spatial structure of the larvae. Presence of non-sporulating cadavers was also observed, which may be as a result of within-host competition. Fungal outgrowth after host death was extremely limited in many dual-inoculated cadavers, and it is possible that in the nonsporulating cadavers a high concentration of each pathogen led to mutual antagonisms or scramble competition where neither of the isolates survived. Limited sporulation or no sporulation of fungus from P. xylostella larvae in a proportion of cadavers infected by P. blunckii isolate NW449 alone has been reported previously

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(Guzma´n-Franco 2005). Although this suggests that isolate NW449 results in non-sporulating cadavers or cadavers with limited sporulation even when not competing with Z. radicans, the proportion of this type of cadaver was much greater in dual-inoculated larvae, suggesting that scramble competition was occurring between P. blunckii isolate NW449 and the Z. radicans isolates during vegetative growth. This was confirmed by the fact that cadavers resulting from dual-inoculation with a different P. blunckii isolate, NW454, and the Z. radicans isolates also produced cadavers with limited sporulation or no sporulation at all. Pandora blunckii isolate NW454 when inoculated alone onto P. xylostella larvae always resulted in sporulating cadavers (Guzma´n-Franco 2005). Similar results in microsporidia have been described as a result of scramble competition (Solter et al. 2002). Fargues & Bon (2004) also found non-sporulating cadavers during dual-inoculation with two isolates of Paecilomyces fumosoroseus (Wise) Brown & Smith on G. mellonella larvae, but their results were more related to a temperature effect than a concentration effect. Krauss et al. (2004) found non-sporulating cadavers as a result of dual-inoculation with one entomopathogenic fungus–either Beauveria bassiana (Balsamo) Vuillemin or Metarhizium anisopliae (Metschnikoff) Sorokin–and one mycoparasite–either Clonostachys sp. or Trichoderma harzianum Tul. They found more non-sporulating cadavers in small insects–Bemisia tabaci (Gennadius)–than in large insects–Sitophilus oryzae L.–which suggested that larger insects may have provided more protection against negative competition in internal habitats which were not accessible to the mycoparasite. Although Z. radicans isolate NW386 was the least virulent of all the four isolates (Guzma´n-Franco 2005), the relationship between NW386 and the two P. blunckii isolates was similar to that described for Z. radicans isolate NW250, which supported the hypothesis that interactions between Z. radicans and P. blunckii are related to the initial concentration ratio of each pathogen. However, a significant relationship was found between Z. radicans isolate NW386 and the proportion of dual-infected larvae. This suggests that this isolate may have stronger competitive abilities than Z. radicans isolate NW250, because NW250 could only compete effectively with both P. blunckii isolates if its initial conidial inoculation concentration was larger than its competitor. However, NW386 had the ability to survive even when it was outnumbered in conidia at inoculation because it produced morphologically identifiable resting spores inside cadavers. The greater ability of Z. radicans isolate NW386 to survive dual-inoculations (through the production of resting spores) with the more virulent P. blunckii isolate NW449 may be the result of co-evolution between these isolates. When dual-inoculations of P. xylostella larvae were made in Petri dishes, both pathogens had the same opportunity to infect the larvae. However, when the experiment was done on cabbage plants, inoculation was passive. This experiment was carried out on a larger spatial scale than the Petri dish experiment, and the way that the larvae became infected was also similar to the way that they may be infected in the field if we consider a sporulating plug as a simulation of a sporulating cadaver. Larvae were infected as a consequence of

A. W. Guzma´n-Franco et al.

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being closer to the inoculum, but could also receive inoculum at different times, increasing the spatial and temporal heterogeneity considerably. However, under these conditions, the results obtained showed a very similar pattern to those obtained in Petri dishes. The presence of P. blunckii isolate NW449 reduced the likelihood of both Z. radicans isolates infecting P. xylostella larvae and vice versa. This may be because one cabbage plant did not provide sufficient spatial heterogeneity to allow each fungus species to perform differently. The overall proportions of infection were smaller than in the Petri dish experiments, which suggest that not all the larvae contacted the fungi, and, therefore, did not receive a sufficient concentration to initiate infection (Regoes et al. 2002) or no conidia at all. Although the experimental conditions were optimal for the fungi throughout, the results are important because they provide an insight into what may be happening in the field. In the field, conditions are not favourable for the fungi all the time, but there are always some periods of optimal conditions for both fungal species, otherwise no infected cadavers would be found at all. If both species can co-exist on a plant, they will be more likely to co-exist and survive at even larger spatial scales (e.g., brassica crop) and may differentiate niches and avoid competition. Under field conditions, there are many factors that will have an effect on the outcomes of an interaction. Temperature is one of the most important (e.g., Inglis et al. 1996; Fargues & Bon 2004), and also the presence of other microorganisms that may not be direct competitors for the host but could interfere with the infection and survival of the pathogen (Hughes & Boomsma 2004; Krauss et al. 2004; Rosta´s & Hilker 2003). Under field conditions, it has been suggested that two different pathogens may survive by having different distributions or alternate hosts (Perlman & Jaenike 2001; Koppenho¨fer & Kaya 1996). Z. radicans is often considered to be a generalist pathogen compared to P. blunckii, which appears to be a specialist. Z. radicans may, therefore, infect other insect species than P. xylostella to survive and thereby not compete directly with P. blunckii, but this remains to be tested experimentally with these isolates (e.g., Pell et al. 2001; Glare et al. 1987). The proportion of cadavers with UCM was larger than in the Petri dish experiment, but again it showed a significant interaction with the presence of P. blunckii isolate NW449. However, these large proportions of cadavers with UCM were also present in the controls. This may have been the result of the high humidity conditions resulting from use of an unventilated cage. In conclusion, we cannot be certain whether a single isolate was dominant in these interaction experiments. However, our results suggest that isolates of both species can co-exist in the same geographical area, and that outcomes will depend more on the relative concentration of conidia inoculated than on the relative virulence of the isolates. It is possible that the ability of Z. radicans isolate NW386 to produce resting spores gives this isolate an ecological advantage in the long term, and that it can survive direct competition with P. blunckii isolates. The results also suggest that P. blunckii isolates, particularly NW449, are better intra-host competitors than the Z. radicans isolates evaluated.

Acknowledgements AGF was supported by Asociacio´n Nacional de Universidades e Instituciones de Educacio´n Superior (ANUIES) and Colegio de Postgraduados, Mexico. J.K.P. was supported by the Department for Environment, Food and Rural Affairs of the United Kingdom (Defra). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.

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