Mycovirus effect on the endophytic establishment of the entomopathogenic fungus
Tolypocladium cylindrosporum in tomato and bean plants
Noemí Herrero Asensio, Salud Sánchez Márquez, Iñigo Zabalgogeazcoa
Instituto de Recursos Naturales y Agrobiología de Salamanca, IRNASA-CSIC.
Cordel de Merinas 40-52, 37008 Salamanca, Spain. e-mail: i.zabalgo@irnasa.csic.es
Abstract
Two endophytic strains of the entomopathogenic fungus Tolypocladium cylindrosporum , originally isolated from the grass Festuca rubra , were artificially inoculated in tomato and bean plants. Strains 11-1L and 11-0BR were isolated from asymptomatic leaf fragments of both plant species at 3, 7, 14, 21, and 35 days after their inoculation. The percentage of leaf fragments infected by the fungus in inoculated leaves decreased at each sampling time, and no systemic colonization of the plants occurred. The two T. cylindrosporum strains tested were isogenic, differing in the infection by the Victorivirus
TcV1, harboured by strain 11-1L, but not by 11-0BR. The percentage of infected leaf fragments in leaves inoculated with the virus-infected strain was greater in bean than in tomato plants, while the virus-free strain was more successful in tomato than in bean plants.
This result suggests that the mycovirus infection can affect the adaptation of T. cylindrosporum to particular host plants.
Key words: biological control, dsRNA, mycovirus, multitrophic interactions
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Introduction
Endophytes are fungi capable of infecting plant tissues without causing any apparent symptoms to their plant hosts. Surveys done in numerous plant species have shown that the presence of this type of fungi is normal in plants, and a large number of fungal species can behave as endophytes (Arnold 2007; Hyde and Soytong 2008; Sánchez Márquez et al. 2012).
Several roles have been attributed to some endophytic species, like increasing the tolerance of their host plants to abiotic stressors, or protection against plant pathogenic fungi, nematodes, and herbivorous insects (Clay and Schardl 2002; Rodríguez et al. 2009; Vega et al. 2008;
Zabalgogeazcoa 2008). However, the outcome of the symbioses between most endophytic fungi and their hosts remains unknown.
Species belonging to entomopathogenic genera like Beauveria Vuill., Isaria Pers.,
Cladosporium Link, Metarhizium
Sorokïn
, Lecanicillium W. Gams and Zare , or
Tolypocladium W. Gams , have been reported as naturally occurring endophytes in different plant species (Bills and Polishook 1990; Miles et al. 2012; Sánchez Márquez et al. 2012;
Vega et al. 2008). Although most research on entomopathogenic fungi has been focused in developing inundative methods for their use as biological control agents of insects, mites, and ticks, the discovery of their endophytic nature is considered as a new approach for the use of entomopathogenic fungi in agriculture (Vega et al. 2009). For example, the endophytic capability of B. bassiana (Bals.-Criv.) Vuill.
, one of the best known mycoinsecticides, has been demonstrated with the successful artificial inoculation of diverse plant hosts using concentrated suspensions of conidia (Biswas et al. 2012; Gómez-Vidal et al. 2006; Ownley et al. 2008; Quesada-Moraga et al. 2006; Tefera and Vidal 2009; Wagner and Lewis 2000). As an endophyte, B. bassiana protected plants against insect herbivores (Bing and Lewis 1991;
Quesada Moraga et al. 2009). Furthermore, some works suggest that the presence of
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endophytic B. bassiana might also protect plants against some fungal and bacterial plant pathogens (Miles et al. 2012; Ownley et al. 2010). In relation to the mode of action of these endophytic entomopathogens, different studies indicate that they may influence the population dynamics of insect herbivores, attributing reduction of damages caused by them to an accumulation of mycotoxins in endophyte-infected plant tissues (Gurulingappa et al. 2011;
Leckie et al. 2008; Vega et al. 2008), or to the direct infection of insects by epiphytic spores produced by the endophytes (Anderson et al. 2007).
Tolypocladium cylindrosporum W. Gams is an entomopathogenic fungus that has been isolated as an endophyte from grasses like Festuca rubra and Holcus lanatus (Lam et al.
1988; Sánchez Márquez et al.
2012). Endophytic T. cylindrosporum strains have been reported to harbour three different mycoviruses, TcV1, TcV2 and TcV3 (Herrero and
Zabalgogeazcoa 2011). However, the presence of these fungal viruses did not affect the pathogenicity of T. cylindrosporum strains to two species of argasid ticks (Herrero et al.
2011). Relationships between fungal viruses and their hosts resemble those of fungal endophytes and plants; mycoviruses rarely cause obvious symptoms on their hosts. Only a few cases of mycoviruses affecting their host fitness are known. Fungal viruses cause hypovirulence in some plant pathogens like Cryphonectria parasitica , and a severe disease in mushrooms like Agaricus bisporus (Ghabrial and Suzuki 2010; Nuss 2005; Romaine and
Goodin 2002). On the other hand, the presence of a mycovirus in the root endophyte
Curvularia protuberata improves the thermal tolerance of the plant host of this endophyte
(Marquez et al.
2007).
The main objective of this work was to test if endophytic strains of T. cylindrosporum originally isolated from grasses, could be inoculated in two plant species of agricultural interest, tomato ( Solanum lycopersicum ) and bean ( Phaseolus vulgaris ), establishing a first step to evaluate the possible use of endophytic T. cylindrosporum as a biocontrol agent. The
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two T. cylindrosporum strains used in the bioassay were isogenic, differing only in the presence of the mycovirus TcV1 (Herrero and Zabalgogeazcoa 2011). Therefore, another objective of this work was to investigate if the mycovirus infection affected the capability of the fungus to behave as an endophyte in tomato and bean plants.
Materials and Methods
Fungal strains and inoculum preparation
T. cylindrosporum strains 11-1L and 11-0BR were conidial progeny of the endophytic strain 11, obtained from a plant of the grass F. rubra . Strain 11 was infected by three different viruses, TcV1, TcV2, and TcV3. This fungal strain was treated with the antiviral compound ribavirin, and several conidial progeny infected by single viruses, or virus free were obtained
(Herrero and Zabalgogeazcoa 2011). This was the case of strain 11-1L, infected only by
TcV1, and strain 110BR, free of the three viruses. These two strains can be considered as isogenic, differing only in the presence of the virus TcV1. There are no significant differences in the culture morphology or radial growth in vitro of these two strains at 22ºC, but at 29ºC the growth of the virus free strain was significantly greater than that of the strain infected by
TcV1 (Herrero 2011). The genome of TcV1 has been completely sequenced, and this virus was identified as a member of the Victorivirus genus, in the Totiviridae family (Herrero and
Zabalgogeazcoa 2011).
To obtain conidial suspensions for plant inoculations, strains were grown in Petri plates containing potato dextrose agar (PDA) at room temperature (22–25 ºC) in the dark.
Conidia from 20 day old cultures were released from the mycelium with a glass rod, after adding 5 ml of sterile water containing 0.001% Tween 80 to each plate. The conidial
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suspensions from the plates were collected and centrifuged at 5000 x g for 10 min. The pellets were resuspended in sterile water, and the concentration of conidia was estimated with a
Bürker chamber. On average we obtained about 1.5 x 10 7
spores from each gram of fresh mycelium. A 20 day old culture from a Petri plate with PDA contained about 1.5 g of mycelium. Suspensions of 1 x 10
8
conidia/ml in water containing 0.001% Tween 80 were used for the inoculations.
Plant inoculations and determination of endophytic infection
Seeds of bean cv. Blanca Planchada and tomato cv. Tres Cantos, were surface sterilised with a 2 min immersion in commercial bleach (5% active chlorine), and rinsed three times with sterile water. Individual seeds were germinated in 16 cm diameter pots filled with a 1:1(v:v) mixture of peat and perlite, which was previously sterilized in an oven at 100 ºC for
72 hours. The tomato and bean plants were grown in a greenhouse at temperatures that oscillated from 14 to 27 ºC between night and day, and at ambient humidity. Tomato plants were 30 days old, and bean plants 15 days old at the time of their inoculation. Both species were inoculated in the same day, and using the same batches of conidia.
Two ml of the conidial suspension (1 x 10
8 conidia/ml in water containing 0.001%
Tween 80) or of the sterile 0.001% Tween 80 control solution were applied to each tomato or bean plant by spraying with a manual atomizer. The four first true leaves of tomato plants, or the first two true leaves of bean plants, were sprayed with the conidial suspension. Twenty five plants of each species were inoculated with each fungal strain, and another 25 plants of each species were mock inoculated with the control solution.
Infection by the two isogenic strains of T. cylindrosporum , 11-1L and 11-0BR, in bean and tomato plants was determined at 3, 7, 14, 21 and 35 days post inoculation (dpi), by fungal
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reisolation. To estimate the amount of infection per plant, small square leaf pieces measuring about 5 mm per side, were cut from the inoculated leaves of each of five plants per treatment at each sampling date. The leaf pieces from each plant were surface sterilized with a solution of 20% commercial bleach (1% active chlorine) for 10 minutes, and rinsed in sterile water.
Thirty two leaf fragments randomly chosen from the excess of pieces cut from each plant sampled at each time interval were placed in two Petri plates (sixteen leaf pieces per plate) containing PDA with 200 mg/l chloramphenicol, and the Petri plates were incubated at room temperature (22–25 ºC) in the dark. The growth of T. cylindrosporum from each leaf piece was recorded up to 30 days after the sampling date, and the number of infected pieces from each sample of 32 pieces was used as an estimate of the amount of infection of the plant from which the sample was taken. The same procedure was followed with the control plants.
To check for the systemic movement of the fungi, tomato and bean leaves formed after the inoculation were processed for the reisolation of T. cylindrosporum at 21 and 35 dpi. In this case, only one PDA plate containing sixteen pieces of leaves from each plant species and treatment were analyzed. Stems and roots were also checked for T. cylindrosporum infection at 35 dpi. One plate containing sixteen pieces of root or stem from each plant was analyzed for each plant species and fungal treatment. Stem fragments were processed as leaves, but root pieces were surface-disinfected by means of a 5 minute rinse with ethanol, followed by treatment with a 1% active chlorine solution for 15 minutes, 2 minutes in ethanol, and a final rinse in sterile water (Bills 1996).
To test if the surface disinfection methods used for leaf, stem, and root pieces were effective eliminating surface fungi, at each sampling date imprints of surface disinfected leaf, stem, and root fragments inoculated with each fungal strain were made in the surface of PDA in Petri plates which were incubated without plant parts (Schulz et al. 1998). The plates were periodically observed to determine if fungi emerged from the imprints. The absence of
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mycelium growing from the imprints indicated that the disinfection methods used were adequate to eliminate epiphytic conidia or hyphae of T. cylindrosporum and other fungi.
Morphological characteristics were used to identify T. cylindrosporum colonies, since all the mycelia emerging from each piece of plant was individually plated in 5.5 cm diameter
Petri dishes containing PDA. Additionally, fungal identification was completed with the molecular identification of five isolates from the total of those reisolated at each sampling time by means of the nucleotide sequence of their ITS1-5.8S rRNA-ITS2 region (Herrero et al. 2011).
Statistical analysis
Differences in the percentage of infected leaf pieces between both fungal treatments and host plant species at each sampling interval (3, 7, 14, 21 and 35 dpi) were analysed using a three-way ANOVA. The control treatment was not included in the statistical analysis because no colonies of T. cylindrosporum were recovered from the mock inoculated plants.
Before the analysis, the normality of the percentage of infected leaf pieces data for each date and host was tested using a Kolmogorov-Smirnov test. P values smaller than 0.05 were considered statistically significant. Statistica 5.0 software (StatSoft, Tulsa, USA) was used to perform these calculations.
Results
During the experiment all inoculated plants looked as healthy as the controls, which did not show any symptom of disease or stress. Both isogenic strains of T. cylindrosporum ,
11-1L, infected by TcV1, and virus-free 11-0BR could be recovered from surface-disinfected
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inoculated leaves of both plant species up to 35 days after the inoculation period (Fig. 1).
Tolypocladium mycelium started to emerge from pieces of tomato and bean leaves about eight days after plating the leaf fragments in plates containing PDA (Fig. 2). Except for 18 isolates of other endophytic species, all reisolated fungi were identified morphologically and molecularly as T. cylindrosporum.
The characteristic white mycelium of Tolypocladium did not emerge from any leaf fragment of the control plants mock inoculated with an aqueous
0.001% Tween 80 solution.
No statistically significant effect of the presence of the mycovirus TcV1 in the infection efficiency of the fungal strains was detected (F
1, 80
= 0.41; p= 0.52). Similarly, the mean percentage of infected leaf pieces was not significantly different between bean or tomato plants (F
1, 80
= 1.17; p= 0.28). However, a significant interaction between plant species and fungal strains was detected (F
1, 80
= 6.00; p= 0.02). This result indicates that the virusinfected strain was more effective infecting bean leaves than the virus-free strain, but the virus-free strain was more effective infecting tomato leaves than its virus infected version
(Fig. 1). No other significant interactions were detected by the ANOVA.
Significant differences in the percentage of infected leaf pieces occurred at the different sampling dates (F
4, 80
= 30.79; p<0.001). In bean and tomato plants, the percentage of infected leaf pieces decreased as sampling dates post inoculation increased (Fig. 1). However, in both species the decrease in the recovery of the fungus over time was more linear for strain
11-0BR than for the TcV1 infected strain. At 14 dpi the recovery of strain 11-L1 was greater than at 7 dpi in both plant species (Fig. 1).
Very few infected fragments were detected in the experiments made to test the possible systemic movement of the fungus, indicating that this is unlikely to occur. In new leaves of tomato formed after the inoculation, infection was detected in four of 160 fragments at 21dpi, and the same number was observed in bean. No endophytic infections were detected
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in new leaves at 35 dpi in either host. The low number of infections detected might be the result of experimental errors like the inoculation of leaf primordia at the initial inoculation time, or leaf surface infections by inoculum from the surface of inoculated leaves being transported to new leaves. Furthermore, in tomato stems Tolypocladium was recovered only from three out of 160 stem pieces, and in bean from one out of 160. No infected root fragments were detected in either host.
Discussion
Several species of entomopathogenic fungi have been found to have a naturally occurring endophytic phase in their life cycles (Vega 2008; Vega et al. 2009; Wagner and
Lewis 2000). This is also the case of T. cylindrosporum , which has been isolated from wild plants of the grasses Festuca rubra and Holcus lanatus (Sánchez Márquez et al. 2012). In this study we show the endophytic establishment of T. cylindrosporum in bean and tomato plants following conidial applications. The fact that the two strains tested in the bioassay can behave as endophytes in bean and tomato, species different from their original grass host, indicates the multihost capability of this fungus as an endophyte. This is a desirable characteristic for a potential biocontrol agent, because it makes possible its application on different crop species.
In contrast with our result, an endophytic strain of a Tolypocladium spp. isolated from cocoa plants could not be successfully reinoculated in its original host (Hanada et al. 2010).
The time course followed after the inoculation showed that the presence of the fungus in plants decreases over time. This occurred in both plant species, and is in concordance with results observed in inoculations of B. bassiana , L. lecanii or Aspergillus parasiticus in different crops (Gurulingappa et al. 2010; Posada et al, 2007). Declines in percentage colonization over time have been attributed to a defensive response from the plant, the
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establishment of localized colonies in the leaves, or competition with other endophytes
(Gurulingappa et al. 2010; Posada et al, 2007). A current model of plant-endophyte interactions proposes that endophytism is the result of situations of “balanced antagonism”, where the fungus can modulate host defense reactions, and the plant fungal growth (Schulz and Boyle, 2005).
Our results do not demonstrate the systemic movement of the fungus in bean or tomato plants. A very small number of infected fragments from newly formed leaves and stems were observed, and these could be the result of external infections by conidia instead of systemic colonization by the fungus. In contrast to the behaviour of T. cylindrosporum , B. bassiana has been shown to move systemically in plant hosts (Cherry et al. 1999; Quesada-Moraga et al.
2006). Further studies could be made in order to test if other inoculation methods would result in the systemic colonization of plants by T. cylindrosporum . Other inoculation techniques, like dressing seeds with conidia, dipping roots or rhizomes in conidial suspensions, injecting conidia in rhizomes or stems, or even applying conidia to plant substrates, have been succesfully used with B. bassiana (Akello et al. 2007; Gómez-Vidal et al. 2006; Posada and
Vega 2006; Quesada-Moraga et al. 2006; Tefera and Vidal 2009).
The presence of the virus TcV1 seemed to affect the progress of the infection by the T. cylindrosporum strains. A significant virus x host interaction suggested that the virus-free strain 11-0BR was more effective as an endophyte in bean than in tomato, in contrast, the virus infected strain 11-1L was more effective in tomato than in bean. This could indicate that the changes induced by the virus in the fungus might affect the fungal adaptation to new hosts. Another possible effect of the presence of the virus is the difference between strains observed at 14 dpi, when the presence of the fungus increased for the virus infected strain in both host plants.
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Beauveria bassiana strains originally obtained from insects have been shown to have endophytic capability (Posada and Vega 2005, 2006; Quesada Moraga et al. 2006; Tefera and
Vidal 2009). In this work we show that T. cylindrosporum strains originally obtained as grass endophytes can also infect other plant species. In addition, the strains used in this study are derived from endophytic strain 11, which was shown to be pathogenic against two different species of ticks (Herrero et al. 2011). These relationships show how entomopathogens have developed intricate interactions with arthropods and plants. Increasing knowledge of the ecology of entomopathogens is crucial for understanding their role in natural and managed ecosystems, and for their successful development as biocontrol agents (Cory and Ericsson
2010; Roy et al. 2010; Vega et al. 2009). Here we add another player to the tritrophic interactions in which entomopathogenic fungi are involved, mycoviruses, which seem to affect the ability of Tolypocladium to colonize different hosts. More studies in this topic would increase the understanding of the ecological interactions established by entomopathogenic fungi, improving their efficacy as biological control agents.
In conclusion, this study demonstrates that T. cylindrosporum strains originally isolated as endophytes from the grass Festuca rubra , were able to establish an endophytic relationship with other plant species than its original host. Therefore, T. cylindrosporum could have a potential as biological control agent of invertebrate herbivores or even for the control of plant pathogens, as have been proposed for other entomopathogenic fungi (Ownley et al.
2010). T. cylindrosporum has several characteristics that make it suitable for this purpose, like having a wide plant host range, being pathogenic to several insect taxa, and to other arthropods; as well as its abundant sporulation, and the viability of those spores after storage
(Herrero et al. 2011). This study also shows evidence of an effect of a virus in the endophytic behaviour of a fungus.
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Figures
Fig. 1 Percentage of infected leaf fragments of bean ( a) and tomato ( b ) leaves at 3, 7, 14, 21 and 35 days post inoculation with a virus infected (11-1L) and a virus free (11-0BR) strain of
Tolypocladium cylindrosporum . Error bars indicate the standard error of the mean.
70 a
60
50
40
30
20
10
0
3
 11-1L
 11-0BR
7 14 21 days post inoculation
35
40
30
20
10
0
70 b
60
50
3


11-1L
11-0BR
7 14 21 days post inoculation
35
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Fig. 2 a Petri plate with pieces of bean leaves which were sprayed 21 days before with a fungal suspension of 1 x 10
8
conidia/ml showing growth of T. cylindrosporum strain 11-1L. b
Hyphae of strain 11-0BR emerging from a piece of bean leaf inoculated 21 days before.
(a) (b)
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