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Endophytic blastospores of Beauveria bassiana provide high resistance against plant disease caused by Botrytis cinerea
Abstract
The entomopathogenic fungus Beauveria bassiana is widely used for insect pest control and can produce three distinct infective unit types under different nutritional and environmental conditions: aerial conidia, blastospores, and submerged conidia. Here, we identified endophytic colonization ability and existing forms of the three types of B. bassiana infective units after inoculating Arabidopsis plants via soil drenching, and tested their effects on their presence mold disease caused by Botrytis cinerea. We found that all B. bassiana infective unit types colonized Arabidopsis leaves, with germinating and producing hyphae by hydrophilic blastopores and submerged conidia; further, we showed that blastospores were more effective in defending against B. cinerea, compared with aerial conidia. These findings suggest that in addition to aerial conidia, the colonization of other two types of entomopathogenic fungal infective units could also have important impacts on plant resistance. This study contributes to better understanding on the function of B. bassiana as fungal endophytes, which could lead to a new paradigm for how to successfully use these organisms in biological control against plant diseases.
... Endophytic B. bassiana reportedly suppresses Rhizoctonia solani and Pythium myriotylum growth in tomato (Ownley et al., 2004) and cotton (Ownley et al., 2008), Zucchini yellow mosaic virus colonization in squash (Jaber and Salem, 2014), and Plasmopara viticola colonization in grapevines (Jaber, 2015). Despite the substantial evidence available on the antagonistic activity of endophytic EPF against phytopathogens (Ownley et al., 2010;Sui et al., 2022), such activity remains understudied when compared with their recognized biocontrol activity against insect pests. Investigation of the biocontrol activity of B. bassiana against phytopathogens, and elucidating its mode of action against disease-causing organisms, could enhance the biopesticide potential of the fungus greatly. ...
... Other researchers have confirmed B. bassiana colonization in plants using scanning electron microscopy and molecular biology techniques, providing a convenient method for further determination of its endogenous activity (Landa et al., 2013;Behie et al., 2015). Recently, the distribution characteristics of B. bassiana in maize plant tissues have been clarified using green fluorescent protein (GFP)-labeled strains, which has provided technical support for further exploration of endophytic bacteria colonization in plant tissues (Sui et al., 2022). In summary, numerous studies have demonstrated that B. bassiana in plant tissues is symbiotic; however, a clear understanding of the preferential localization within plant tissues is still lacking. ...
... The pathogen often infects leaves, stems, flowers, and fruits of the host plants (Williamson et al., 2007). Sui et al. (2022) observed that compared with aerial conidia, hydrophilic blastopores of B. bassiana more effectively defend against B. cinerea. Although the positive effects of EPF in plants have been documented extensively (Vega, 2018;Quesada-Moraga, 2020), whether and how EPF influence plant resistance to biotic stress remains largely unknown. ...
Introduction
Entomopathogenic fungi (EPF) can colonize and establish symbiotic relationships with plants as endophytes. Recently, EPF have been reported to suppress plant pathogens and induce plant resistance to diseases. However, the potential mechanisms via which EPF as endophytes control major plant diseases in situ remain largely unknown.
Methods
Pot and field experiments were conducted to investigate the mechanisms via which an EPF, Beauveria bassiana , colonizes tomato, under Botrytis cinerea infection stress. B. bassiana blastospores were inoculated into tomato plants by root irrigation. Tomato resistance to tomato gray mold caused by B. cinerea was evaluated by artificial inoculation, and B. bassiana colonization in plants and rhizosphere soil under B. cinerea infection stress was evaluated by colony counting and quantitative PCR. Furthermore, the expression levels of three disease resistance-related genes ( OXO , CHI , and atpA ) in tomato leaves were determined to explore the effect of B. bassiana colonization on plant disease resistance performance in pot experiments.
Results
B. bassiana colonization could improve resistance of tomato plants to gray mold caused by B. cinerea . The incidence rate, lesion diameter, and disease index of gray mold decreased in both the pot and field experiments following B. bassiana colonization. B. bassiana was more likely to accumulate in the pathogen infected leaves, while decreasing in the rhizosphere soil, and induced the expression of plant resistance genes, which were up-regulated in leaves.
Discussion
The results indicated that plants could “recruit” B. bassiana from rhizosphere soil to diseased plants as directional effects, which then enhanced plant growth and resistance against pathogens, consequently inhibiting pathogen infection and multiplication in plants. Our findings provide novel insights that enhance our understanding of the roles of EPF during pathogen challenge.
... In addition to the infective potential of these propagules, recent research has highlighted the capacity of blastospores and submerged conidia to endophytically colonize plants. This colonization can subsequently trigger plants' defense mechanisms against phytopathogens more efficiently when compared to aerial conidia (Sui et al. 2022). ...
... According to the literature, submerged conidia have an intermediate germination speed between aerial conidia and blastospores (Jaronski and Mascarin 2017), so it was not possible to observe a higher rate of infection in treatments with SS. Blastospores are characterized by a longer cylindrical shape (5-8 µm), while submerged conidia have a smaller size, in the range of 3-5 µm (Sui et al. 2022), which characterizes the largest portion of SS observed for IBCB 868 and CBMAI 1306 strains. ...
In this study, IBCB 66, IBCB 868, and CBMAI 1306 isolates of Beauveria bassiana were grown in liquid culture for 4 days, leading to elevated submerged spores (SS) levels. The influence of the addition of different glycerol concentrations (0, 3, and 6%) (v/v) in the liquid culture was investigated regarding the stability (at 4 and 27 °C) of dried formulations. The virulence of SS was compared with aerial spores (AS) against Tetranychus urticae (Koch) (Acari: Tetranychidae). The results demonstrate the potential of using SS to control T. urticae. CBMAI 1306 and IBCB 868 isolates caused T. urticae mortality rates of 91.11% and 88.89% 5 days after treatment, respectively, when applied at concentrations of 1 × 10⁸ SS mL⁻¹. The median Lethal Time (LT50) values for these strains were 2.64 and 2.61 days, respectively. The dried formulations showed potential acaricidal activity. Higher glycerol concentrations in the liquid culture medium reduced formulation stability at 27 °C.
... 39,40 Our findings align with previous research that demonstrated the ability of B. bassiana hypha to penetrate and colonize within plant tissue. 41,42 This process may wileyonlinelibrary.com/journal/ps affect the mechanisms of plant defense against insect pests. ...
BACKGROUND
Biological control of insect pests is encountering an unprecedented challenge in agricultural systems due to the ongoing rise in carbon dioxide (CO2) level. The use of entomopathogenic fungi (EPF) in these systems is gaining increased attention, and EPF as crop endophytes hold the potential for combining insect pest control and yield enhancement of crops, but the effects of increased CO2 concentration on this interaction are poorly understood. Here, the introduction of endophytic EPF was explored as an alternative sustainable management strategy benefiting crops under elevated CO2, using maize (Zea mays), Asian corn borer (Ostrinia furnacalis), and EPF (Beauveria bassiana) to test changes in damage to maize plants from O. furnacalis, and the nutritional status (content of carbon, nitrogen, phosphorus, potassium), biomass, and yield of maize.
RESULTS
The results showed that endophytic B. bassiana could alleviate the damage caused by O. furnacalis larvae for maize plants under ambient CO2 concentration, and this effect was enhanced under higher CO2 concentration. Inoculation with B. bassiana effectively counteracted the adverse impact of elevated CO2 on maize plants by preserving the nitrogen content at its baseline level (comparable with ambient CO2 conditions without B. bassiana). Both simultaneous effects could explain the improvement of biomass and yield of maize under B. bassiana inoculation and elevated CO2.
CONCLUSION
This finding provides key information about the multifaceted benefits of B. bassiana as a maize endophyte. Our results highlight the promising potential of incorporating EPF as endophytes into integrated pest management strategies, particularly under elevated CO2 concentrations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
... The incidence rate of soybean leaves was observed and recorded at 4, 5, 6, and 7 days post-inoculation (dpi); the disease incidence was calculated as follows: incidence rate (%) = (number of incidents/total number of inoculated plants) × 100 (Li et al., 2023). The diameter of disease spots on diseased leaves in each treatment was calculated by the cross method at 4 dpi (Sui et al., 2022). ...
Introduction
Aspergillus nomiae is known as a pathogenic fungus that infects humans and plants but has never been reported as an entomophagous fungus (EPF) that can provide other functions as an endotype.
Methods
A strain of EPF was isolated and identified from diseased larvae of Spodoptera litura in a soybean field and designated AnS1Gzl-1. Pathogenicity of the strain toward various insect pests was evaluated, especially the ability to colonize plants and induce resistance against phytopathogens and insect pests.
Results
The isolated EPF strain AnS1Gzl-1 was identified as A. nomiae; it showed strong pathogenicity toward five insect pests belonging to Lepidoptera and Hemiptera. Furthermore, the strain inhibited the growth of Sclerotinia sclerotiorum in vitro, a causal agent of soil-borne plant disease. It colonized plants as an endophyte via root irrigation with a high colonization rate of 90%, thereby inducing plant resistance against phytopathogen infection, and disrupting the feeding selectivity of S. litura larvae.
Discussion
This is the first record of a natural infection of A. nomiae on insects. A. nomiae has the potential to be used as a dual biocontrol EPF because of its ability to not only kill a broad spectrum of insect pests directly but also induce resistance against phytopathogens via plant colonization.
... Endophytic fungi are capable of colonizing into host plants through various ways, thereby enhancing their growth [31][32][33]. Endophytes leveraged to date include Beauveria bassiana [34][35][36][37][38], Metarhizium anisopliae [39][40][41], Isaria fumosorosea [42,43], M. robertsii [44,45], and Paecilomyces lilacinus [46,47]. These fungi have been used to colonize corn [32], the common bean [48], arabidopsis [49], tomatoes [50], wheat, lemen [51], vicia faba [52], lettuce [53], tobacco [22] and other crops [51] via root irrigation [54], seed soaking [32], seed coating [55], stem base injection [56], root or rhizome soaking [46], leaf spraying [56], root mixing, and other methods [57]. ...
The use of entomogenous fungi as endophytes is currently an area of active research. Isaria cateniannulata is an important entomogenous fungus that has been employed for the active control of a range of pests in agricultural and forestry settings, but its direct impact on plants remains to be evaluated. Herein, we assessed the ability of I. cateniannulata to colonize buckwheat, Fagopyrum esculentum and F. tataricum, and its impact on buckwheat defense enzyme activity and physiological indexes. The majority of fungal submerge condia was able to enter into leaves through stomata and veins, and this was followed by conidial attachment, lytic enzyme secretion, conidial deformation, and enhanced defensive enzyme activity within buckwheat, followed by the repair of damaged tissue structures. I. cateniannulata populations on buckwheat leaf surfaces (in CFU/g) reached the minimum values at 24 h after inoculation. At this time, the blast analysis revealed that the sequence identity values were 100%, which was consistent with the sequence of I. cateniannula. The number of I. cateniannulata submerge conidia colonized in the buckwheat leaves gradually rose to peak levels on 7 d post-inoculation, and then gradually declined until 10 d, at which time the buckwheat plant growth index values increased. This study provided novel evidence that I. cateniannulata could be leveraged as an endophytic fungus capable of colonizing buckwheat plants and promoting their growth.
BACKGROUND
Entomophagous fungi (EPF) not only directly kill insect pests, but also colonize plants and improve their resistance against pests. However, most previous research has focused on Beauveria bassiana and Metarhizium anisopliae, and there are few reports on whether other EPF can enhance resistance against pests via endogenous colonization. Herein, an EPF strain was isolated from diseased larvae of Spodoptera litura in a soybean field, and subjected to genome‐wide sequencing at the chromosomal level. The pathogenicity of the isolate toward various pest insects was evaluated, and the ability to colonize plants and induce resistance against phytopathogens and insect pests was tested.
RESULTS
The purified isolate was identified as M. rileyi and designated MrS1Gz1–1. Biological assays revealed its strong pathogenicity toward five insect pests belonging to Lepidoptera and Hemiptera. Furthermore, the strain inhibited the growth of soil‐borne plant disease caused by Sclerotinia sclerotiorum in vitro. It colonized plants as an endophyte via soil application, thereby inducing plant resistance‐related genes against phytopathogen infection, and it disrupted the feeding selectivity of S. litura larvae.
CONCLUSION
M. rileyi MrS1Gz1–1 has potential as a broad‐spectrum microbial control agent that can induce resistance against phytopathogens and insect pests feeding as an endotype. The complete genome provides a valuable resource for exploring host interactions. © 2024 Society of Chemical Industry.
Systemic crop protection using endophytic isolates of entomopathogenic fungi is at the forefront of IPM. Nonetheless, any potential trade-offs between virulence and endophytic behaviour must be elucidated if they are to be effectively used in pest management strategies. Here we investigated endophytic adaptation in an isolate of Beauveria bassiana following successive passage through melon, tomato and cotton tissues. Plants were sprayed with a suspension of B. bassiana endophytic isolate EABb 04/01-Tip to initiate endophytic colonization. Once colonization was established, the fungus was re-isolated from the plant, applied to another plant and re-isolated again; this was repeated to achieve three passages. After each passage, a conidial suspension of each isolate was used in bioassays to evaluate both virulence against 4th instar larvae of the model insect Galleria mellonella and to quantify the extent of endophytic activity in each respective host plant species. When sprayed leaves were inspected for fungal colonization, differences in percentage tissue colonization amongst the plant species were detected after the first re-isolation. Endophytic colonization rates in melon and tomato, which varied from 70-100%, were higher than those observed in cotton, which ranged from 40-50%; endophytic colonization in cotton increased to 75-100% after the third passage. This improvement in endophytic behaviour in cotton, an apparently suboptimal plant for fungal colonization, suggests an evolutionary adaptation to localized or transient endophytic colonization, while further assays are needed. Meanwhile, when endophytic colonization of non-sprayed leaves distant from the sprayed ones was investigated, endophytic activity was evident in all three crop species suggesting that movement within plants after successive passage increased the extent of endophytic colonization from transient to systemic. The present research highlights the potential for adaptation to endophytic behaviour in crops that are less suitable for endophytic colonization. Furthermore, we demonstrated stability in virulence after multiple passages through host plants. This is a key result for the development of IPM strategies based on endophytic entomopathogenic fungi.
BACKGROUND
It has been suggested that entomopathogenic fungi can be introduced into plants as endophytes potentially leading to insect control. Here, we sought to identify specific strains of the insect pathogenic fungus, Beauveria bassiana that would form endophytic associations with tobacco (Nicotiana benthamiana) benefitting host plant growth and/or resistance against insect pests and pathogens.
RESULTS
Tobacco seeds were inoculated with six different B. bassiana strains and entophytic colonization, plant growth, and resistance to pathogens and insect pests were evaluated over a 50 day‐period. Although all the strains could colonize seedlings, 90% seedling colonization was seen for four strains. Fungal cells could be detected in stems more readily than in leaf and root tissues. Colonization by B. bassiana boosted plant growth with an increased photosynthetic rate, chlorophyll content, and stomatal and trichome density seen in fungal treated plants. Tobacco seedlings colonized by specific B. bassiana strains displayed significantly increased tolerance/resistance against bacterial and fungal pathogens. B. bassiana‐colonized seedlings also displayed higher resistance to aphids (Myzus persicae) as compared to untreated controls. Colonization by B. bassiana was shown to trigger both of the salicylic acid (SA) and jasmonate acid (JA) defense pathways, but SA pathway was upregulated much more than JA pathway for some of the tested strains.
CONCLUSION
Specific strains of B. bassiana can be introduced into host plants as endophytes, resulting in promotion of host plant growth, increased resistance to microbial pathogens, and/or increased resistance to insect pests. © 2020 Society of Chemical Industry
The possible effects of climate change on species interactions remain a very complex and challenging subject in community ecology. Here, we comparatively examine the interactions between maize (Zea mays) and an endophytic fungal entomopathogen (Beauveria bassiana) in a typical agroecosystem under both ambient and elevated air temperatures. We found that under ambient temperature certain key biological characteristics in maize (i.e., height, relative growth rate, biomass, and defense enzymes) and B. bassiana (i.e., conidia yield, germination rate of conidia, and virulence) were positively related to each other. Under elevated air temperature, however, we only detected positive effects of maize on B. bassiana (i.e., conidia yield, germination rate of conidia, and virulence), but little effect of B. bassiana on maize. These observations suggest that elevated air temperature could shift the interactions between plants and the endophytic fungal entomopathogens, possibly even from mutualism to commensalism. Both the nature and strength of species interactions are important for better understanding and predicting structure and stability of ecological communities in agroecosystems under climate change.
Beauveria bassiana, known for its entomopathogenic characteristics, is the most widely used biocontrol agent against many insect pests and may also be active against soil-borne pathogens. It inhabits the surfaces or inner tissues of various plant species without causing any visible signs or symptoms. Here we show that B. bassiana strain GHA, the active ingredient of a commercial microbial insecticide, colonises tomato plants. GHA grew on intact leaf surfaces of tomato in high humidity, but never entered stomata. Viable hyphae and conidia were detected, and the population on inoculated leaves significantly increased until 14 days after inoculation. On tomato leaves, GHA conidiated normally via conidiophores and phialides, and also via microcycle conidia-tion (conidiophores and phialides form directly from germ tubes and produce conidia). Hyphae were also detected inside the rachis, even more frequently after plant surfaces were scarified. These results suggested that B. bassiana strain GHA can grow epiphytically and endophytically on tomato plants.
Botrytis cinerea causes substantial losses in tomato and chili pepper crops worldwide. Endophytes have shown the potential for the biological control of diseases. The colonization ability of native endophyte strains of Beauveria bassiana and their antifungal effect against B. cinerea were evaluated in Solanaceae crops. Root drenching with B. bassiana was applied, and endophytic colonization capacity in roots, stems, and leaves was determined. The antagonistic activity was evaluated using in vitro dual culture and also plants by drenching the endophyte on the root and by pathogen inoculation in the leaves. Ten native strains were endophytes of tomato, and eight were endophytes of chili pepper. All strains showed significant in vitro antagonism against B. cinerea (30-36%). A high antifungal effect was observed, and strains RGM547 and RGM644 showed the lowest percentage of the surface affected by the pathogen. Native strains of B. bassiana colonized tomato and chili pepper tissues and provided important levels of antagonism against B. cinerea.
An upsurge of research hints at the ability of entomopathogenic fungi, almost exclusively considered and used as insect
pathogens, to endophytically colonize the internal tissues of a wide array of host plants and subsequently confer numerous
benefits including enhancement of plant growth and suppression of disease pathogens. Such an ability has mainly been
investigated for Beauveria bassiana. Fewer studies have demonstrated plant growth promotion by Metarhizium brunneum
colonization, whereas no studies have reported on the potential of endophytic M. brunneum as a plant disease antagonist.
The present study was, therefore, conducted to investigate whether seed treatment with B. bassiana (NATURALIS) and
M. brunneum (BIPESCO5) could result in their endophytic establishment in wheat and promote plant growth. The study
further examines the effect of the fungal strains as endophytes against Fusarium culmorum, one of the main causal agents
of crown and root rot (CRR) in wheat. Both B. bassiana and M. brunneum were able to systemically colonize roots and
shoots of wheat, and promote several plant growth parameters (shoot height, root length, and fresh root and shoot weights).
Moreover, endophytic colonization of wheat with either fungal entomopathogen resulted in a significant reduction in disease
incidence, development and severity. These results support the notion of the multiple ecological roles that could further be
played by entomopathogenic fungi. Bearing such additional roles in mind while developing these fungi as microbial agents
could improve the value of many commercially available mycoinsecticides.
Entomopathogenic fungi from the genus Beauveria (Vuillemin) play an important role in controlling insect populations and have been increasingly utilized for the biological control of insect pests. Various studies have reported that Beauveria bassiana (Bals.), Vuill. also has the ability to colonize a broad range of plant hosts as endophytes without causing disease but while still maintaining the capacity to infect insects. Beauveria is often applied as an inundative spore application, but little research has considered how plant colonization may alter the ability to persist in the environment. The aim of this study was to investigate potential interactions between B. bassiana and Zea mays L. (maize) in the rhizosphere following inoculation, in order to understand the factors that may affect environmental persistence of the fungi. The hypothesis was that different isolates of B. bassiana have the ability to colonize maize roots and/or rhizosphere soil, resulting in effects to the plant microbiome. To test this hypothesis, a two-step nested PCR protocol was developed to find and amplify Beauveria in planta or in soil; based on the translation elongation factor 1-alpha (ef1α) gene. The nested protocol was also designed to enable Beauveria species differentiation by sequence analysis. The impact of three selected B. bassiana isolates applied topically to roots on the rhizosphere soil community structure and function were consequently assessed using denaturing gradient gel electrophoresis (DGGE) and MicroResp TM techniques. The microbial community structure and function were not significantly affected by the presence of the isolates, however, retention of the inocula in the rhizosphere at 30 days after inoculation was enhanced when plants were subjected to intensive wounding of foliage to crudely simulate herbivory. The plant defense response likely changed under wound stress resulting in the apparent recruitment of Beauveria in the rhizosphere, which may be an indirect defensive strategy against herbivory and/or the result of induced systemic susceptibility in maize enabling plant colonization.
Fungal entomopathogens have been proposed as environmentally friendly alternatives to chemical control. Unfortunately, their effectiveness continues to be limited by their susceptibility to ultraviolet (UV) light and low moisture. A relatively recent development, the use of fungal entomopathogens as endophytes, might overcome the traditional obstacles impeding the widespread adoption of fungal entomopathogens and also provide a novel alternative for management of insect pests and plant pathogens. In addition, some fungal entomopathogens could also function as biofertilizers. Eighty-five papers covering 109 individual fungal entomopathogen studies involving 12 species in six genera are reviewed. Thirty-eight plant species in 19 families were studied, with maize, common bean, and tomato being the most investigated. Of the 85 papers, 39 (46%) examined the effects of fungal entomopathogen endophytism on 33 insect species in 17 families and eight orders. Thirty-four (40%) examined plant response to endophytism, corresponding to 20 plant species. Various inoculation techniques (e.g., foliar sprays, soil drenching, seed soaking, injections, etc.) are effective in introducing fungal entomopathogens as endophytes, but colonization appears to be localized and ephemeral. The field of insect pathology will not substantially profit from dozens of additional studies attempting to introduce fungal entomopathogens into a wider array of plants, without attempting to understand the mechanisms underlying endophytism, the responses of the plant to such endophytism, and the consequent responses of insect pests and plant pathogens. This review presents several areas that should receive focused attention to increase the probability of success for making this technology an effective alternative to chemical control.
Two experimental replicates were conducted to test whether strains
of Beauveria brongniartii (BIPESCO2 and 2843) and Metarhizium
brunneum (BIPESCO5) can endophytically colonise Vicia faba
plants and improve their growth by comparing them with an
endophytic strain of B. bassiana (NATURALIS®). The plants were
inoculated through foliar spray and the effect of inoculation on
plant height, leaf pair number, fresh root and shoot weights was
measured at 7 and 14 days post inoculation (dpi). Endophytic
colonisation of different plant parts with the tested fungal strains
were confirmed 7 and 14 dpi through re-isolation of inoculated
fungi onto selective media and subsequent Simple Sequence
Repeat (SSR) marker-based genetic identification. All tested strains
were able to endophytically colonise leaves, stems, and even roots
of inoculated plants 7 and 14 dpi, but per cent colonisation varied
significantly among strains and plant parts within each sampling
date. Foliar inoculation of plants with the tested strains increased
plant height, leaf pair number, fresh shoot and root weights;
however the increase was not always consistent across sampling
dates in both experimental replicates. This study provides the first
evidence for the endophytic colonisation of plants with two
strains of B. brongniartii, an important biocontrol agent of
Melolontha melolontha and other scarab beetles in several
European countries, and thus extends previous reports on the
ability of entomopathogenic fungi to act as endophytes. It also
presents possible explanations for the lack of consistency in the
plant growth promotion obtained by the foliar inoculation of
entomopathogenic fungi.
We investigated the fungal entomopathogens Beauveria bassiana and Metarhizium anisopliae to determine if endophytic colonization could be achieved in cassava. An inoculation method based on drenching the soil around cassava stem cuttings using conidial suspensions resulted in endophytic colonization of cassava roots by both entomopathogens, though neither was found in the leaves or stems of the treated cassava plants. Both fungal entomopathogens were detected more often in the proximal end of the root than in the distal end. Colonization levels of B. bassiana were higher when plants were sampled at 7-9 days post-inoculation (84%) compared to 47-49 days post-inoculation (40%). In contrast, the colonization levels of M. anisopliae remained constant from 7-9 days post-inoculation (80%) to 47-49 days post-inoculation (80%), which suggests M. anisopliae is better able to persist in the soil, or as an endophyte in cassava roots over time. Differences in colonization success and plant growth were found among the fungal entomopathogen treatments.
The filamentous fungus Beauveria bassiana is an economically important pathogen of numerous arthropod pests and is able to grow in submerged culture as filaments (mycelia) or as budding yeast-like blastospores. In this study, we evaluated the effect of dissolved oxygen and high glucose concentrations on blastospore production by submerged cultures of two isolates of B. bassiana, ESALQ1432 and GHA. Results showed that maintaining adequate dissolved oxygen levels coupled with high glucose concentrations enhanced blastospore yields by both isolates. High glucose concentrations increased the osmotic pressure of the media and coincided with higher dissolved oxygen levels and increased production of significantly smaller blastospores compared with blastospores produced in media with lower concentrations of glucose. The desiccation tolerance of blastospores dried to less than 2.6 % moisture was not affected by the glucose concentration of the medium but was isolate dependent. Blastospores of isolate ESALQ1432 produced in media containing 140 g glucose L−1 showed greater virulence toward whitefly nymphs (Bemisia tabaci) as compared with blastospores produced in media containing 40 g glucose L−1. These results suggest a synergistic effect between glucose concentration and oxygen availability on changing morphology and enhancing the yield and efficacy of blastospores of B. bassiana, thereby facilitating the development of a cost-effective production method for this blastospore-based bioinsecticide.
A study was conducted to examine whether Beauveria
bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) can colonize grapevine leaf tissues and subsequently confer protection against downy mildew caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Following the foliar inoculation of plants with conidial suspensions of selected B. bassiana strains, colonization of leaves by the fungus was determined using culture-based and PCR techniques at different time intervals. Seven days following B. bassiana inoculation, grapevine plants were challenged with P. viticola and symptoms were assessed by calculating the disease incidence and severity. Although all tested strains were able to colonize grapevine plants, percent colonization differed significantly among strains. Disease incidence and severity were, on the other hand, significantly reduced in B. bassiana-inoculated plants compared to control plants irrespective of strain. This study is one of very few studies investigating the promising role B. bassiana could play as a plant disease antagonist.
Brazil and China have been successful in the use of microbial control methods to manage several agricultural and forest insects. In both countries, entomopathogenic fungi (EF) have been used for pest management since the 1970s. However, EF production and commercialization have not been constant in either country. Several companies and cooperatives suspended their activities or shut down from the 1970s to the 1990s. This was due to loss of confidence in available mycoinsecticides by Brazilian farmers or due to reduced involvement and government subsidies for biological control in China; and, consequently, mycoinsecticides were largely replaced by inexpensive chemical insecticides. Starting in the 1990s and continuing until today, however, new Brazilian and Chinese private companies have arisen. In Brazil, the area treated with M. anisopliae for spittlebug control alone is estimated to be approximately one million hectares in 2008, 75% of which was for control of spittlebugs in sugarcane plantations and the remainder for spittlebugs in pasture grass (primarily Brachiaria spp.) and other smaller programs. In China, the fungus Beauveria bassiana was used annually in 0.8–1.3 million ha until the 1980s. Several factors were important for the success of these programs, such as: governmental support (at least during the initial steps of biocontrol programs); availability of indigenous virulent fungal isolates; low-cost substrates for mass production; retail prices of mycoinsecticides lower than their chemical counterparts; and sale by contract which allows the products to be immediately available for use, rather than stored. In this report, we discuss the current biocontrol programs using insect fungi in these two developing countries, as well as the future and main challenges they must face to further encourage the adoption of mycoinsecticides.
Under submerged growth in a defined medium (TKI broth), the entomopathogenic fungus, Beauveria bassiana, produced conidia; it produced only blastospores in complex media. Production of such "submerged" conidia depended on the nature of the carbon source and the presence of nitrate as a nitrogen source. Maximum yield of conidia (5 × 108 mL) was obtained when glucose was the carbon source and when the glucose to nitrate ratio was 5:1. Other carbon sources gave rise to both conidia and blastospores. Reducing the phosphate concentration resulted in the production of conidia which resembled "aerial" conidia in morphology and germination rates. The surfaces of "submerged" conidia were relatively smooth, but had a tendency to acquire the rough, warty, brittle surface characteristics of aerial conidia. Blastospores produced in defined media gave rise to conidia through microcycle conidiation without going through the vegetative phase of growth. In more complex media, blastospores did not undergo microcycle conidiation.
A study was conducted to determine the effect of inoculation method and plant growth medium on colonization of sorghum by
an endophytic Beauveria bassiana. Colonization of leaves, stems, and roots by B. bassiana was assessed 20-days after application of the fungus. Although B. bassiana established as an endophyte in sorghum leaves, stems, and roots regardless of inoculation method (leaf, seed, or soil inoculation),
plant growth medium (sterile soil, non-sterile soil, or vermiculite) apparently influenced colonization rates. Seed inoculation
with conidia caused no stem or leaf colonization by the fungus in non-sterile soil but did result in substantial endophytic
colonization in vermiculite and sterile soil. Leaf inoculation did not result in root colonization, regardless of plant growth
medium. Endophytic colonization was greater in leaves and stems than roots. Endophytic colonization by B. bassiana had no adverse effects on the growth of sorghum plants. Leaf inoculation with a conidial suspension proved to be the best
method to introduce B. bassiana into sorghum leaves for plants growing in either sterile or non-sterile soil. Further research should focus on the virulence
of endophytic B. bassiana against sorghum stem borers.
An important mechanism for insect pest control should be the use of fungal entomopathogens. Even though these organisms have been studied for more than 100 y, their effective use in the field remains elusive. Recently, however, it has been discovered that many of these entomopathogenic fungi play additional roles in nature. They are endophytes, antagonists of plant pathogens, associates with the rhizosphere, and possibly even plant growth promoting agents. These findings indicate that the ecological role of these fungi in the environment is not fully understood and limits our ability to employ them successfully for pest management. In this paper, we review the recently discovered roles played by many entomopathogenic fungi and propose new research strategies focused on alternate uses for these fungi. It seems likely that these agents can be used in multiple roles in protecting plants from pests and diseases and at the same time promoting plant growth.
The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.
The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.
Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ
tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on
the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located,
indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal
structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed
within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities
for biological control, including the use of indigenous fungal inocula as insecticides.
The attachment of the conidia of the insect-pathogenic fungi Nomuraea rileyi, Beauveria bassiana, and Metarrhizium anisopliae to insect cuticle was mediated by strong binding forces. The attachment was passive and nonspecific in that the conidia adhered readily to both host and nonhost cuticle preparations. The hydrophobicity of the conidial wall and the insect epicuticle appeared to mediate the adhesion process. Detergents, solvents, and high-molecular-weight proteins known to neutralize hydrophobicity reduced conidial binding when added to conidium-cuticle preparations. However, these chemicals did not remove the hydrophobic components from the epicuticle or from conidial preparations. The outer surface of the conidium consists of a resilient layer of well-organized fascicles of rodlets. Intact rodlets extracted from B. bassiana conidia bound to insect cuticle and exhibited the hydrophobicity expressed by intact conidia. Both electrostatic charges and various hemagglutinin activities were also present on the conidial surface. However, competitive-inhibition studies indicated that these forces played little, if any, role in the adhesion process.
The entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and ∼2500 5′ end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1079 contigs, respectively. Almost half (40-45 %) of the sequences in each library displayed either no significant similarity (e value > 10-4) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2416 unique sequences, of which 20-30 % were unique to each library, and only ∼6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental conditions.
Marked differences in surface characteristics were observed among three types of single-cell propagules produced by the entomopathogenic fungus Beauveria bassiana. Atomic force microscopy (AFM) revealed the presence of bundles or fascicles in aerial conidia absent from in vitro blastospores and submerged conidia. Contact angle measurements using polar and apolar test liquids placed on cell layers were used to calculate surface tension values and the free energies of interaction of the cell types with surfaces. These analyses indicated that the cell surfaces of aerial conidia were hydrophobic, whereas those of blastospores and submerged conidia were hydrophilic. Zeta potential determinations of the electrostatic charge distribution across the surface of the cells varied from +22 to -30 mV for 16-day aerial conidia at pH values ranging from 3 to 9, while the net surface charge ranged from +10 to -13 mV for submerged conidia, with much less variation observed for blastospores, +4 to -4 mV, over the same pH range. Measurements of hydrophobicity using microbial adhesion to hydrocarbons (MATH) indicated that the surfaces of aerial conidia were hydrophobic, and those of blastospores hydrophilic, whereas submerged conidia displayed cell surface characteristics on the borderline between hydrophobic and hydrophilic. Insect pathology assays using tobacco budworm (Heliothis virescens) larvae revealed some variation in virulence among aerial conidia, in vitro blastospores and submerged conidia, using both topical application and haemocoel injection of the fungal cells.
Fungi that occur inside asymptomatic plant tissues are known as fungal endophytes. Different genera of fungal entomopathogens have been reported as naturally occurring fungal endophytes, and it has been shown that it is possible to inoculate plants with fungal entomopathogens, making them endophytic. Their mode of action against insects appears to be due to antibiosis or feeding deterrence. Research aimed at understanding the fungal ecology of entomopathogenic fungi, and their role as fungal endophytes, could lead to a new paradigm on how to successfully use these organisms in biological control programs.
Cordyceps, comprising over 400 species, was historically classified in the Clavicipitaceae, based on cylindrical asci, thickened ascus apices and filiform ascospores, which often disarticulate into part-spores. Cordyceps was characterized by the production of well-developed often stipitate stromata and an ecology as a pathogen of arthropods and Elaphomyces with infrageneric classifications emphasizing arrangement of perithecia, ascospore morphology and host affiliation. To refine the classification of Cordyceps and the Clavicipitaceae, the phylogenetic relationships of 162 taxa were estimated based on analyses consisting of five to seven loci, including the nuclear ribosomal small and large subunits (nrSSU and nrLSU), the elongation factor 1alpha (tef1), the largest and the second largest subunits of RNA polymerase II (rpb1 and rpb2), beta-tubulin (tub), and mitochondrial ATP6 (atp6). Our results strongly support the existence of three clavicipitaceous clades and reject the monophyly of both Cordyceps and Clavicipitaceae. Most diagnostic characters used in current classifications of Cordyceps (e.g., arrangement of perithecia, ascospore fragmentation, etc.) were not supported as being phylogenetically informative; the characters that were most consistent with the phylogeny were texture, pigmentation and morphology of stromata. Therefore, we revise the taxonomy of Cordyceps and the Clavicipitaceae to be consistent with the multi-gene phylogeny. The family Cordycipitaceae is validated based on the type of Cordyceps, C. militaris, and includes most Cordyceps species that possess brightly coloured, fleshy stromata. The new family Ophiocordycipitaceae is proposed based on Ophiocordyceps Petch, which we emend. The majority of species in this family produce darkly pigmented, tough to pliant stromata that often possess aperithecial apices. The new genus Elaphocordyceps is proposed for a subclade of the Ophiocordycipitaceae, which includes all species of Cordyceps that parasitize the fungal genus Elaphomyces and some closely related species that parasitize arthropods. The family Clavicipitaceaes. s. is emended and includes the core clade of grass symbionts (e.g., Balansia, Claviceps, Epichloë, etc.), and the entomopathogenic genus Hypocrella and relatives. In addition, the new genus Metacordyceps is proposed for Cordyceps species that are closely related to the grass symbionts in the Clavicipitaceaes. s.Metacordyceps includes teleomorphs linked to Metarhizium and other closely related anamorphs. Two new species are described, and lists of accepted names for species in Cordyceps, Elaphocordyceps, Metacordyceps and Ophiocordyceps are provided.
Endophytes are recently gaining interest because they can offer novel forms of biological pest control. Endophytic growth of arthropod pathogenic fungi can parasitize insect herbivores without causing damage to the crop. However, studies addressing this tritrophic interaction are absent. Here, the most known endophytic arthropod pathogenic fungal species Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordyciptaceae), the polyphagous two-spotted spider mite Tetranychus urticae Koch (Trombidiformes: Tetranychidae), and its preferred plant host Phaseolus vulgaris L. (Fabales: Fabaceae) were selected to study the multi-kingdom interactions among plants, arthropods, and entomopathogenic fungi. T. urticae were reared on non-colonized and endophytically colonized tissues of P. vulgaris. Real-Time PCR analysis of nine defense-related genes revealed that a broad range of plant defense mechanisms is activated in response to the endophytic growth of B. bassiana. Moreover, we studied the molecular mechanism adapted by the two-spotted spider mite that underlies resistance. The analysis of 41 detoxification genes revealed that relatively moderate, high, and few numbers of genes were changed in the adults, nymphs, and eggs stages of T. urticae, respectively, after inoculation on colonized tissues of P. vulgaris. The endophytic growth of B. bassiana can have a negative effect on the growth and performance of the pest, in a developmental stage-dependent manner, by priming plant defense pathways. In parallel, the herbivore induces a broad range of detoxification genes that could potentially be involved in adaptation to endophytically colonized plant tissues.
This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, isolated locally was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions using readily available agricultural residues. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of B. bassiana SAN01 xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present other promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.
Traditionally, the soil has been considered the main reservoir of entomopathogenic mitosporic ascomycetes (AMEs) together with the insect populations that they regulate, whereas mostly during the XXI century, the association of AMEs with the plants playing additional ecological roles, in the rhizosphere, in phylloplane, and as plant endophytes has been highlighted. The endophytic behaviour of AMEs has modified the basis behind their use as biocontrol agents in agriculture providing new approaches and delivery methods to pest control and crop production. Several studies have focused on the use of fungal endophytic strains as microbial control agents for systemically protecting crops against cryptic pests, whose life cycle seriously limits the viability of chemicals and other control techniques. Noteworthy, endophytic EPF may allow not only the protection of the plant against boring, chewing and sucking pests, but they can improve the plant response to other biotic (i.e. plant diseases) and abiotic stresses (i.e. nutritional), with promotion of plant health and plant growth being also demonstrated. Contrary to traditional mycoinsecticides, the utilisation of AMEs as endophytes has the benefit of contacting the pest inside the plant at diminished application costs since little inoculum is required. Moreover, the endophytic fungal biocontrol agent is shielded within the plant from hindering abiotic and biotic factors that would restrict its utilisation as an epiphyte. The present work on broader contribution of AMEs to IPM has been carried out at least partially from the experience of the research group AGR 163 “Agricultural Entomology” of the University of Cordoba (Spain).
Blastospores are yeast-like infective cells to arthropod pests and can be obtained shortly by liquid culture. However, fungal isolates exhibit striking variation in production, desiccation tolerance and virulence. In this study, we screened three isolates of Beauveria bassiana (Balsamo) Vuillemin and three of Cordyceps fumosorosea (Wize) (Hypocreales: Cordycipitaceae) for blastospore production and assessed desiccation tolerance by air drying. The virulence of these isolates were determined against Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B, Spodoptera frugiperda (J.E. Smith) and Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae). The isolates ESALQ-1296 and ESALQ-3422 of C. fumosorosea and ESALQ-4958 of B. bassiana yielded blastospore concentrations above 1 × 109 ml−1, but only the two C. fumosorosea isolates retained viability rates above 75% after drying. Blastospores of both fungal species were more effective in killing the three insect species than aerial conidia. In summary, blastospores of C. fumosorosea ESALQ-1296 hold the best promise for broad-spectrum pest control in soybean.
Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists—mainly for technical reasons—on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea. Indeed, in contrast to previous reports, most OGs were acetyl- and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors.
Entomopathogenic fungi are promising alternatives to synthetic chemicals for controlling lesser mealworm populations in broiler houses. Granular formulations of two isolates of the fungi Beauveria bassiana and Metarhizium anisopliae based on ground chicken feed pellets produced 98 and 87% larval mortality, respectively, after 11 days’ exposure to ≈ 0.2 g conidia/54 g formulation m⁻² in the laboratory. Oil based formulations with canola oil and Codacide™ were generally slower to produce mortality but still significantly effective. Granular formulations were effective on a range of broiler-house substrates (new and used litter, soil) of varying pH and moisture content. The efficacy of the M. anisopliae isolate appeared enhanced and the B. bassiana isolate inhibited on damp new litter (40% moisture). Substrate pH (3.45–6.38) did not discernibly inhibit fungal efficacy. Granular formulations of both fungal isolates tested at average broiler-house temperatures were ineffective at 35 °C but became active when re-tested at 30 °C. Granular mycoinsecticide formulations based on the two isolates are now ready for testing in commercial broiler houses.
The entomopathogenic fungus Beauveria bassiana is able to colonize and establish a symbiotic relationship with plants as an endophyte, naturally as well as through artificial inoculation. This study aims at implementing a reliable and efficient method to establish endophytic B. bassiana in maize plants. The level of the endophytic establishment was evaluated and the survival and fecundity of the English grain aphid (Sitobion avenae) was investigated. Eight-days-old maize plants were sprayed with a fungal spore suspension, while control plants were treated only with a Tween solution. Two weeks after inoculation, seven clip cages per plant, each containing an adult aphid, were attached to different leaves and left for seven days. All inoculated plants contained endophytic B. bassiana as confirmed by plating surface disinfected leaf pieces on selective plates. None of the control plants showed growth of B. bassiana. The highest colonization levels were 61% in older inoculated leaves and 19% in younger non-inoculated leaves. The survival of S. avenae on inoculated plants was reduced up to 49% as compared to control plants. Dead aphids did not show any signs of mycosis. The number of nymphs produced by a single aphid was reduced up to 70% and 74% on inoculated and non-inoculated leaves, respectively. Presence of endophytic B. bassiana in non-inoculated younger leaves implies the movement of the endophyte inside plants. The negative effect on aphid fecundity appears to be systemic as implied by the almost equal reduction of mean numbers of nymphs produced on older and younger leaves on the inoculated plants, irrespective of the level of colonization by endophytic B. bassiana.
In the present study we investigated the infection process of Beauveria bassiana on Xylotrechus rusticus larvae using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM results showed that B. bassiana spores germinated on the surface of the larval body and invaded the larva as an appressorium. The hyphae then germinated from the spores and spread throughout the larval body. After the death of the larva, conidiophores formed at one end of the hypha on the surface of the larval body and prepared for a new round of infection. The TEM results showed severe damage to the larval cuticle after hyphae infection. The structure of the cuticle became thinner and eventually flocculent; muscle tissues were dissociated and eventually stuck to the hyphae, and the corpus adiposum was loose and deformed, and eventually degraded.
An increasing number of recent studies demonstrate that entomopathogenic fungi, often solely considered as insect pathogens, play additional roles in nature, including endophytism, plant disease antagonism, plant growth promotion, and rhizosphere colonization. These newly emerging, but not yet fully understood, ecological roles hint at the possibility that we have been overlooking important attributes in our quest to develop fungal entomopathogens exclusively as inundative biopesticides against insect and other arthropod pests. Such additional roles recently-discovered to be played by entomopathogenic fungi provide opportunities for the multiple use of these fungi in integrated pest management (IPM) strategies. Of particular interest is the ability displayed by various genera of entomopathogenic fungi to colonize a wide variety of plant species in different families, both naturally and artificially following inoculation, and confer protection against not only insect pests but also plant pathogens. This article reviews the literature currently available on the endophytic colonization of different host plants by fungal entomopathogens, and summarizes the negative effects of such colonization on insect pests and plant pathogens that have been reported to date. It also addresses the possible mechanisms of protection conferred by endophytic fungal entomopathogens and explores the potential use of these fungi as dual microbial control agents against both insect and pathogen pests. Moreover, interactions amongst endophytic fungal entomopathogens and other endophytes are discussed. Finally, current limitations and future research directions for the innovative use of endophytic fungal entomopathogens as dual microbial control agents are summarized.
Pest insects cause severe damage to global crop production and pose a threat to human health by transmitting diseases. Traditionally, chemical pesticides (insecticides) have been used to control such pests and have proven to be effective only for a limited amount of time because of the rapid spread of genetic insecticide resistance. The basis of this resistance is mostly caused by (co)dominant mutations in single genes, which explains why insecticide use alone is an unsustainable solution. Therefore, robust solutions for insect pest control need to be sought in alternative methods such as biological control agents for which single-gene resistance is less likely to evolve. The entomopathogenic fungus Beauveria bassiana has shown potential as a biological control agent of insects, and insight into the mechanisms of virulence is essential to show the robustness of its use. With the recent availability of the whole genome sequence of B. bassiana, progress in understanding the genetics that constitute virulence towards insects can be made more quickly. In this review we divide the infection process into distinct steps and provide an overview of what is currently known about genes and mechanisms influencing virulence in B. bassiana. We also discuss the need for novel strategies and experimental methods to better understand the infection mechanisms deployed by entomopathogenic fungi. Such knowledge can help improve biocontrol agents, not only by selecting the most virulent genotypes, but also by selecting the genotypes that use combinations of virulence mechanisms for which resistance in the insect host is least likely to develop.
This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000
Arabidopsis thaliana L. has many features favoring its
use as a model in studies of plant-growth-promoting rhizobacteria (PGPR), such
as diazotrophs. Several niches are colonized in the root system of
Arabidopsis, including xylem, and intact colonized roots
can be observed microscopically without sectioning of tissues. Studies of
plant genes involved in colonization are facilitated by the ease with which
plants are transformed and by the availability of mutant lines and other
accessions obtainable from stock centers. Lines of
Arabidopsis carrying reporter gene fusions are helping
to reveal the pattern of expression of previously cloned plant genes induced
by rhizobacteria. Studies utilizing indole-3-acetic acid (IAA)-producing PGPR
and Arabidopsis that contain an auxin-responsive GUS
fusion suggest that plants perceive IAA released by bacteria in the
rhizosphere. The role of flavonoids in the colonization of non-legumes is
being assessed using transgenic Arabidopsis with altered
flavonoid metabolism and using tt mutants, which lack
functional versions of specific genes for flavonoid metabolism. Studies of
plant defence and of stress responses are producing molecular data on plant
genes induced by inoculation of Arabidopsis roots with
non-pathogens.
Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae, are environmentally friendly biocontrol agents (BCAs) against various arthropod pests. We provide an overview to the past-decade advances in fungal BCA research and application in China. Since 1960s, fungal BCAs have been mass-produced for application and at present, thousands of tons of their formulations are annually applied to control forest, agricultural, greenhouse and grassland insect pests throughout the country. Apart from technical advances in mass production, formulation and application of fungal BCAs, basic studies on the genomics, molecular biology, genetic engineering and population genetics of fungal entomopathogens have rapidly progressed in the past few years in China. The completed genomic studies of M. anisopliae, Metarhizium acridum, B. bassiana and Cordyceps militaris provide profound insights into crucial gene functions, fungal pathogenesis, host–pathogen interactions and mechanisms involved in fungal sexuality. New knowledge gained from the basic studies has been applied to improve fungal virulence and stress tolerance for developing more efficacious and field-persistent mycoinsecticides by means of microbial biotechnology, such as genetic engineering. To alleviate environmental safety concerns, more efforts are needed to generate new data not only on the effects of engineered BCAs on target and non-target arthropods but also on their potential effects on gene flow and genetic recombination before field release.
Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH4Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 105/ ml after two days in GlcNAc medium as compared to 8.9 105/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 106/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.
Beauveria bassiana can produce three spore types; aerial conidia, submerged conidia and blastospores. We have examined the spore surface characteristics (hydrophobicity and cell-wall surface lectins), thermal inactivation and the virulence towards the migratory grasshopper, Melanoplus sanguinipes, of each of the three spore types. The hydrophobicities of the aerial and submerged conidia were quite similar. Blastospores were less hydrophobic than either of the two types of conidia. Hydrophobic interactions are thought to play a significant role in attachment of the spore to the host organism. However, the less hydrophobic blastospores were slightly more virulent (LT50 of 6.50 days) when compared to the aerial and submerged conidia (7.12 and 7.24 days), respectively. The lectin-binding characteristics of the aerial and submerged conidia were very similar but differed from that of blastospores. Growth of blastospores on a variety of carbohydrates did not affect their lectin-binding characteristics. Spore viability measurements showed that aerial and submerged conidia retained their viability for a longer period than blastospores. The similarity in hydrophobicity, stability, virulence and lectin-binding of aerial and submerged conidia make the latter an ideal candidate for mycoinsecticide production since they can be recovered after growth on inexpensive substrates.
A thioredoxin (BbTrx) was identified from the entomopathogenic fungus Beauveria bassiana. The cloned nucleotide sequence consisted of a 423-bp open reading frame encoding a 141-amino-acid thioredoxin, a 1011-bp 5' region, and a 419-bp 3' region. The deduced protein sequence of BbTrx, including a common 95-amino-acid conserved domain and a unique 46-amino-acid carboxy terminal region, was similar (≤38% identity) to that of other thioredoxins and phylogenetically closest to that from Neurospora crassa. In insulin solution containing dithiothreitol at 25 °C, recombinant BbTrx or a truncated form lacking the carboxy terminal region (BbTrxD) exhibited disulfide reduction activity. BbTrxD was more active after pre-incubation at 40-75 °C, and cells expressing BbTrxD showed significantly higher tolerance to thermal stress (51 °C). The BbTrx expression in B. bassiana was greatly elevated when stressed at 40 °C. The results indicate that the new thioredoxin is a potential target for improving the thermotolerance of B. bassiana formulations.
Reliable methods for disease severity assessment are of crucial importance in the study of plant pathogen interactions, either for disease diagnostic on the field or to assess phenotypical differences in plants or pathogen strains. Currently, most of the assays used in fungal disease diagnostic rely on visual assessment of the symptoms, lesion diameter measurement or spore counting. However, these tests are tedious and often cannot discriminate between slightly different levels of resistance. Besides, they are not well suited to assess fungal development in the early phases of the infection, before macroscopical symptoms are visible or before sporulation. We describe here a pathogenicity assay based on the relative quantification of fungal and plant DNA in infected Arabidopsis thaliana leaves by means of real-time quantitative PCR. We show that it allows to monitor quantitatively the growth of the fungi Alternaria brassicicola and Botrytis cinerea in a sensitive and reliable way. Although highly sensitive, this test also exhibits a high robustness, which is crucial to significantly discriminate between lines displaying slightly different levels of resistance. Therefore, it allows to assess fungal development from the very first stages of infection and provides a fast and very practical alternative to currently described assays for phenotyping either plant mutant lines or fungal strains.
A novel system was developed for efficient transformation of the fungal biocontrol agent Beauveria bassiana. Competent blastospores were prepared and stored in LiAc- and glycerol-inclusive suspension at -76 degrees C for sequential use in transformation. The system was successfully applied to integrating phosphinothricin resistance gene bar and enhanced green fluorescence protein gene egfp into B. bassiana via blastospore absorption of a plasmid vectoring bar and egfp. A frequency of 24 transformants per microgram of DNA was achieved. The blastospore-based transformation system has proven to be very convenient and would be highly potential for use in genetic manipulation of B. bassiana and other filamentous species.
Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution
- Ownley