Endophytic Fungal Entomopathogens with Activity Against Plant Pathogens: Ecology and Evolution

Home , Digita, Lecanicillium, Lecanicillium lecanii, Lecanicillium muscarium, Podosphaera fuliginea

BioControl (2010) 55:113–128 DOI 10.1007/s10526-009-9241-x

Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution

Bonnie H. Ownley • Kimberly D. Gwinn • Fernando E. Vega

Received: 17 September 2009 / Accepted: 12 October 2009 / Published online: 28 October 2009 Ó International Organization for Biological Control (IOBC) 2009

Abstract Dual biological control, of both insect resistance when endophytically colonized cotton seed- pests and plant pathogens, has been reported for the lings were challenged with a bacterial plant pathogen fungal entomopathogens, Beauveria bassiana (Bals.- on foliage. Species of Lecanicillium are known to Criv.) Vuill. (Ascomycota: Hypocreales) and Lecan- reduce disease caused by powdery mildew as well as icillium spp. (Ascomycota: Hypocreales). However, various rust fungi. Endophytic colonization has been the primary mechanisms of plant disease suppression reported for Lecanicillium spp., and it has been are different for these fungi. Beauveria spp. produce an suggested that induced systemic resistance may be array of bioactive metabolites, and have been reported active against powdery mildew. However, mycopara- to limit growth of fungal plant pathogens in vitro. In sitism is the primary mechanism employed by Lecan- plant assays, B. bassiana has been reported to reduce icillium spp. against plant pathogens. Comparisons of diseases caused by soilborne plant pathogens, such as Beauveria and Lecanicillium are made with Tricho- Pythium, Rhizoctonia, and Fusarium. Evidence has derma, a fungus used for biological control of plant accumulated that B. bassiana can endophytically pathogens and insects. For T. harzianum Rifai (Asco- colonize a wide array of plant species, both monocots mycota: Hypocreales), it has been shown that some and dicots. B. bassiana also induced systemic fungal traits that are important for insect pathogenicity are also involved in biocontrol of phytopathogens.

Handling Editor: Helen Roy. Keywords Beauveria bassiana Á Fungal endophyte Á Hypocreales Á Induced systemic B. H. Ownley (&) Á K. D. Gwinn Department of Entomology and Plant Pathology, resistance Á Lecanicillium Á Mycoparasite Á The University of Tennessee, 2431 Joe Johnson Drive, Trichoderma 205 Ellington Plant Sciences Bldg, Knoxville, TN 37996-4560, USA e-mail: [email protected] Introduction K. D. Gwinn e-mail: [email protected] Resource availability can trigger shifts in functional- F. E. Vega ity within a fungal species, thereby changing the Sustainable Perennial Crops Laboratory, ecological role of the organism (Termorshuizen and United States Department of Agriculture, Jeger 2009). Shifts from one resource to another may Agricultural Research Service, Building 001, BARC-West, Beltsville, MD 20705, USA necessitate significant adaptations in metabolism, e-mail: [email protected] particularly if the resources are dissimilar (Leger 123 114 B. H. Ownley et al. et al. 1997). Among members of the Hypocreales, released by a fungal entomopathogen, carbon source animal, fungal, and plant resources are exploited. played a major role in VOC production by B. These fungi gain nutrition in a variety of ways, bassiana. When cultured on glucose-based media, including: saprotrophs that colonize the rhizosphere the VOCs identified were diisopropyl naphthalenes and phyllosphere, endophytic saprotrophs, hemibio- (\50%), ethanol (ca. 10%) and sesquiterpenes (6%), trophs and necrotrophs of plants, entomopathogens, but in media with n-octacosane (an insect-like and mycoparasites. Some of these fungi function in alkane), the primary VOCs were n-decane (84%) more than one econutritional mode. Fungi tradition- and sesquiterpenes (15%) (Crespo et al. 2008). ally known for their entomopathogenic characteris- Enzymes involved in antibiosis are distinctly tics, such as Beauveria bassiana (Bals.-Criv.) Vuill. different from those involved in mycoparasitism of (Ascomycota: Hypocreales) and Lecanicillium spp. plant pathogens. For example, the biocontrol fungus (Ascomycota: Hypocreales), have recently been Talaromyces flavus Tf1 (Klo¨cker) Stolk & Samson shown to engage in plant-fungus interactions (Vega (Ascomycota: Eurotiales) produces the enzyme glu- 2008; Vega et al. 2008), and both have been reported cose oxidase, whose reaction product, hydrogen to effectively suppress plant disease (Goettel et al. peroxide, kills microsclerotia of phytopathogenic 2008; Ownley et al. 2008). Verticillium (Fravel 1988). Fungal biocontrol organisms actively compete against plant pathogens for niche or infection site, Mechanisms of plant disease suppression carbon, nitrogen, and various microelements. The site by biocontrol fungi of competition is often the rhizosphere, phyllosphere, or intercellularly within the plant. Successful com- Biological control of plant pathogens usually refers to petition is often a matter of timing as resources are the use of microorganisms that reduce the disease- likely to go to the initial colonizer. causing activity or survival of plant pathogens. Mycoparasitism is the parasitism of one fungus by Several different biological control mechanisms another. Varying degrees of host specificity are against plant pathogens have been identified. With displayed by mycoparasites. Within a given species some mechanisms, such as antibiosis, competition, of mycoparasite, some isolates may infect a large and parasitism, the biocontrol organism is directly number of taxonomically diverse fungi, while others involved. With other modes of biological control, demonstrate a high level of specificity (Askary et al. such as induced systemic resistance and increased 1998). As reviewed in Harmon et al. (2004), parasit- growth response, endophytic colonization by the ism by the biocontrol fungus Trichoderma (Ascomy- biocontrol organism triggers responses in the plant cota: Hypocreales) begins with detection of the that reduce or alleviate plant disease. fungal host before contact is made. Trichoderma produces low levels of an extracellular exochitinase, which diffuse and catalyze the release of cell-wall Antibiosis, competition, and mycoparasitism oligomers from the target host fungus. This activity induces Trichoderma to release fungitoxic endoch- The mechanism of antibiosis includes production of itinases, which also degrade the fungal host cell wall. antibiotics, bioactive volatile organic compounds Attachment of the mycoparasite to the host fungus is (VOCs), and enzymes. Volatile bioactive compounds mediated by binding of carbohydrates in the Trich- include acids, alcohols, alkyl pyrones, ammonia, oderma cell wall to lectins in the cell wall of the esters, hydrogen cyanide, ketones, and lipids (Ownley fungal host. Upon contact, hyphae of Trichoderma and Windham 2007). The fungal endophyte Muscodor coil around the host fungus and form appressoria. albus Worapong, Strobel & W.M. Hess (Ascomycota: Several lytic enzymes are involved in degradation of Xylariales) produces a mixture of VOCs that are lethal the cell walls of fungal and oomycetous plant to a variety of microorganisms (Strobel et al. 2001; pathogens, including chitinases, ß-1,3 gluconases, Mercier and Jimeńez 2004; Mercier and Smilanick proteases, and lipases. 2005; Strobel 2006), as well as to insects (Riga et al. In many cases, mechanisms of biocontrol are not 2008; Lacey et al. 2009). In the first report of VOCs mutually exclusive, i.e. multiple mechanisms may be 123 Endophytic fungal entomopathogens 115 operating against a specific plant pathogen, or a given 2009). Induction of systemic resistance via the JA/ biocontrol fungus may employ different mechanisms ethylene signaling pathway has been reported pri- against different phytopathogens. For example, con- marily for plant growth-promoting bacteria, however, trol of Botrytis cinerea Pers. (Ascomycota: Heloti- it is also operative for many mycorrhizal fungi ales) on grapes (Vitis) with Trichoderma involves (Gutjahr and Paszkowski 2009) and biocontrol fungi competition for nutrients and mycoparasitism of (Harmon et al. 2004; Vinale et al. 2008). sclerotia, the overwintering, long-term survival structure of Botrytis. Both mechanisms contribute to suppression of the pathogen’s capability to cause Endophytism by fungal entomopathogens and perpetuate disease (Dubos 1987). Following application to leaves as a preventative, Trichoderma Even though the term ‘‘endophyte’’ has several induced resistance to downy mildew, Plasmopara definitions (Hyde and Soytong 2008), it is widely viticola (Berk. & M.A. Curtis) Berl. & De Toni accepted that endophytes are microorganisms present (Oomycota: Peronosporales), in grape (Perazzolli in plant tissues without causing any apparent symp- et al. 2008). Therefore, it is possible that induced toms. Fungal endophytes are widespread and quite systemic resistance may also play a role in biocontrol diverse in nature (Arnold et al. 2000; Arnold 2007). of Botrytis. Induced resistance to Botrytis, following For example, Vega et al. (2009b) reported 257 unique application of T. harzianum T39 Rifai (Ascomycota: ITS genotypes for fungal endophytes isolated from Hypocreales) to roots and leaves of several ecotypes coffee plants in Hawaii, Mexico, Colombia, and of Arabidopsis thaliana (L.) Heynh. has been Puerto Rico. Infection by fungal endophytes can be reported (Korolev et al. 2008). localized (i.e., not systemic; see Saikkonen et al. 1998 and references therein), and establishing a long- term systemic infection with endophytic fungal Induced systemic resistance entomopathogens that can act against plant pathogens will remain a challenge, and should be the focus of Plants are sessile organisms that must develop a intensive study. complex chemical arsenal in order to withstand biotic Isolation of B. bassiana as a fungal endophyte has and abiotic attack. Colonization of plants with been reported for many plants under natural condi- nonpathogenic fungi and bacteria can lead to induced tions, as well as in plants inoculated using various systemic resistance (ISR) in the host plant. Induced methods (Vega 2008; Vega et al 2008). In contrast to resistance is a plant-mediated biocontrol mechanism the several studies dealing with endophytic Beauveria whereby the biocontrol agent and the phytopathogen spp., only a handful of studies have been conducted do not make physical contact with one another. Plants on endophytic Lecanicillium spp. For example, react to the presence of a pathogen with a rapid Lecanicillium dimorphum (J.D. Chen) Zare & W. expression of defense-related genes. For example, Gams and L cf. psalliotae (Treschew) Zare & W. dramatic cellular changes, characterized by rapid Gams have been introduced as endophytes in date necrotization of lemon (Citrus 9 limon (L.) Burm. f.) palms (Phoenix dactylifera L.) (Go´mez-Vidal et al. fruit exocarp cells were observed in fruit treated with 2006), and L. muscarium strain DAOM 198499 Lecanicillium muscarium DAOM 198499 (Petch) (=Verticillium lecanii (Zimm.) Vie´gas) and L. mus- Zare & W. Gams (formerly Cephalosporium musca- carium strain B-2 have been introduced as endo- rium Petch). Phenolic compounds and phenol oxidase phytes in cucumber (Cucumis sativus L.) roots were both present in reactive cells (Benhamou 2004). (Benhamou and Brodeur 2001; Hirano et al. 2008). In contrast, gene expression changes in plants In cytological investigations of cucumber roots, the infected with beneficial fungi tend to be mild, and the entomopathogen grew actively at the root surface and relationship is allowed to develop resulting in an colonized a small number of epidermal and cortical infected or colonized plant. The signaling mecha- cells, without inducing extensive host cell damage. nisms for this induced resistance are based on Ingress into the root tissue was primarily intercellular jasmonic acid (JA) and ethylene (Van Loon et al. and cell wall penetration was seldom observed 1998; Van Wees et al. 2008; Gutjahr and Paszkowski (Benhamou and Brodeur 2001). Verticillium 123 116 B. H. Ownley et al.

(=Lecanicillium) lecanii has been reported as a 2009a, b). It is likely that more than one mode of natural endophyte in an Araceae (Petrini 1981), in action is operative in suppression of plant disease by Arctostaphylos uva-ursi (L.) (Widler and Mu¨ller B. bassiana. Isolates of the fungus are known to 1984), and in Carpinus caroliniana Walter (Bills produce numerous secondary metabolites (e.g. beau- and Polishook 1991). vericin, beauverolides, bassianolides, oosporein, Although traditionally categorized as a soil sapro- cyclosporin A, and oxalic acid) with antibacterial, phyte, Beauveria spp. are considered to be poor antifungal, cytotoxic, and insecticidal activities competitors for organic resources against other (Grove and Pople 1980; Genthner et al. 1994; Gupta ubiquitous saprophytic soil fungi (Keller and Zim- et al. 1995; Boucias and Pendland 1998; Copping and mermann 1989; Hajek 1997). The endophytic habit of Menn 2000). Effects of these compounds on micro- B. bassiana may provide benefits to both plant and organisms and insects have been reported (Kanaoka fungus. It is well known that plant species has a et al 1978; Taniguchi et al. 1984; Eyal et al. 1994; significant impact on shaping plant-associated micro- Boucias et al. 1995). Recently, another antimicrobial bial communities (Berg et al. 2005; reviewed in Berg compound, bassianolone, from B. bassiana fermen- and Smalla 2009). As suggested by the bodyguard tation culture under low nitrogen conditions, was hypothesis, the plant gains through reduction of characterized (Oller-Lo´pez et al. 2005). Bassianolone damage against herbivorous insects (Elliot et al. has activity against fungi and Gram-positive cocci. 2000; White et al. 2002) or plant diseases; the fungus Antibiosis assays with B. bassiana against various benefits through protection from environmental plant pathogens in vitro have been reported (Table 1). stress, acquisition of limited nutrients from endo- However, the antimicrobial compounds were not phytic colonization as well as exudates on the plant identified. surface, and use of the plant surface as a staging Beauveria bassiana strain 11-98 suppresses plant platform for insect parasitism. On tomato (Solanum disease caused by the soilborne plant pathogens lycopersicum L.) and other dicots, as well as mono- Rhizoctonia solani Ku¨hn (Basidiomycota: Cantharell- cots, colonization by B. bassiana is not restricted to ales) (Ownley et al. 2004) and Pythium myriotylum growth as an endophyte (Ownley et al. 2008; Powell Drechsler (Oomycota: Pythiales) (Clark et al. 2006). et al. 2009; authors, unpublished data). From initial This isolate produces beauvericin (Leckie et al. 2008) establishment as a seed treatment, the fungus can be and oosporein (authors, unpublished data), but it is not found on the outer surfaces as the plant ages, known if these compounds play a role in suppression particularly in areas where new leaves or shoots have of plant disease. Biological control of plant pathogens emerged. The fungus also gains from nutrients with B. bassiana 11-98 is likely to involve competi- acquired during saprophytic colonization of the plant tion for resources (Ownley et al. 2004), since the when it, or parts of it senesce. Similar epiphytic fungus is a plant colonist. Application of B. bassiana growth was observed by Posada and Vega (2005) 11-98 to tomato seed resulted in endophytic and with cocoa (Theobroma cacao L.) seedlings. epiphytic colonization of seedlings and subsequent protection against damping-off. Similarly, seed treatment of cotton (Gossypium hirsutum L.) reduced Beauveria bassiana: Potential for biological severity of R. solani damping-off in seedlings (Griffin control of plant pathogens 2007; Ownley et al. 2008). In both tomato and cotton, the degree of disease control achieved with Beauveria Beauveria bassiana is known to occur naturally in bassiana was correlated with the population density of more than 700 species of insect hosts (Inglis et al. conidia established on seed (Ownley et al. 2008; 2001). Infection of host insects results in the authors, unpublished data). Smaller seeds, such as production of large numbers of conidia, thereby tomato were protected more effectively with rates of serving to increase the population size of the fungus 1 9 106–107 CFU/seed, while higher rates (1 9 107– (Meyling and Eilenberg 2007). There is now 109 CFU/seed) gave the greatest protection against substantial evidence that B. bassiana can provide seedling disease in cotton. protection against some soilborne plant pathogens Parasitism of Pythium myriotylum by B. bassiana (Ownley et al. 2004; Ownley et al. 2008; Vega et al. may be involved in suppression of Pythium damping- 123 Table 1 Studies reporting activity of Beauveria spp. against plant pathogens entomopathogens fungal Endophytic Strain or species of Beauveria Type of study Plant pathogen Activity against plant pathogen Reference

Beauveria bassiana, isolated from In vitro bioassay Gaeumannomyces graminis var. tritici J. Walker Inhibited growth; produced chitinase Renwick et al. wheat rhizosphere In planta (wheat), pot (Ascomycota: Sordariomycetidae) and b-gluconases (1991) assays Suppressed take-all disease Beauveria bassiana (Bals.-Criv.) In vitro bioassay Fusarium oxysporum E.F. Smith & Swingle All Beauveria isolates inhibited Reisenzein and Vuill. (Ascomycota: (Ascomycota: Hypocreales) mycelial growth of the pathogens Tiefenbrunner Hypocreales), five different Armillaria mellea (Vahl) P. Kumm tested (1997) isolates (Basidiomycota: Agaricales) Rosellinia necatrix Berl. ex Prill. (Ascomycota: Xylariales) Culture filtrate of B. bassiana In vitro bioassay Fusarium oxysporum f. sp. lycopersici (Sacc.) W.C. Inhibited mycelial growth; Bark et al. Snyder & H.N. Hansen (Ascomycota Hypocreales) Inhibited and delayed conidial (1996) Botrytis cinerea Pers. (Ascomycota: Helotiales) germination B. bassiana In vitro bioassay Pythium ultimum Trow (Oomycota: Pythiales), Caused cell lysis; inhibited mycelial growth Vesely and B. brongniartii (Sacc.) Petch Pythium debaryanum R. Hesse (Oomycota: Did not inhibit mycelial growth of these Koubova (Ascomycota: Hypocreales) Pythiales), Septoria nodorum (=Phaeosphaeria pathogens (1994) nodorum (E. Mu¨ll.) Hedjar. (Ascomycota: Pleosporales) Rhizoctonia solani Ku¨hn (Basidiomycota: Cantharellales), Pythium irregular Buisman (Oomycota: Pythiales), Phoma betae (=Pleospora betae Bjo¨rl. (Ascomycota: Pleosporales)), Phoma exigua var. foveata Malc. & E.G. Gray (Ascomycota: Pleosporales) Culture filtrates of Beauveria sp. In vitro bioassay Rhizoctonia solani Inhibited mycelial growth; stimulated Lee et al. (1999) growth of cucumber B. bassiana 142, applied to onion In planta (onion), field Fusarium oxysporum f. sp. cepae (Hanzawa) Increased bulb germination; reduced Flori and bulbs and greenhouse W.C. Snyder & H.N. Hansen (Ascomycota: plant infection Roberti (1993) Hypocreales) B. bassiana 11-98, applied In planta (tomato), Rhizoctonia solani Reduced damping off of seedlings; Ownley et al. as a seed treatment greenhouse increased plant growth (2000) and Ownley et al. (2004) B. bassiana 11-98, applied In planta (tomato), Pythium myriotylum Drechsler (Oomycota: Reduced damping off of seedlings Clark et al. as a seed treatment growth chamber Pythiales) (2006) B. bassiana 11-98 In vitro bioassay Rhizoctonia solani Did not inhibit mycelial growth of R. Griffin (2007)

123 B. bassiana 11-98, applied In planta (cotton), Pythium myriotylum solani; but hyphae of 11-98 coiled around and Ownley as a seed treatment growth chamber hyphae of P. myriotylum, which et al. (2008) suggested parasitism Reduced damping-off of seedlings 117 118 B. H. Ownley et al. off in tomato seedlings. In dual culture, hyphae of root) resulted in significantly lower foliar disease isolate 11-98 were observed coiling around the larger ratings for bacterial blight than the untreated control coenocytic hyphae of P. myriotylum (Griffin 2007). and was as effective as 2,6-dichloro-isonicotinic acid, The extent of endophytic colonization of tomato which has been shown to induce systemic resistance by B. bassiana 11-98 was also correlated with the rate against plant pathogens. of conidia applied to seed. Rates that were most effective in disease control also resulted in the greatest degree of plant colonization. Beauveria Lecanicillium spp. and biological control of plant bassiana was detected in root, stem, and leaf sections pathogens of surface-sterilized tomato seedlings with standard dilution plating procedures onto semi-selective med- Lecanicillium spp. (formerly classified in the single ium (Ownley et al. 2008). In addition to seedlings, species Verticillium lecanii) are well known as B. bassiana 11-98 has been recovered from foliage, entomopathogens of aphids and scale insects (Hall stem, and root tissues of surface-sterilized 18-week- 1981; Goettel et al. 2008). These fungi are also old tomato plants produced from treated seed (Powell known as mycoparasites of species of plant patho- et al. 2009). Beauveria bassiana has also been genic, biotrophic powdery mildew (Hall 1980; Ver- recovered as an endophyte of eastern purple cone- haar et al. 1996) and rust fungi (Spencer and Atkey flower (Echinacea purpurea L. Moench), cotton, snap 1981; Allen 1982; Whipps 1993) on various vegeta- bean (Phaseolus vulgaris L.), soybean (Glycines max ble, fruit, and ornamental crops, and as pathogens of L.), and switchgrass (Panicum virgatum L.) following plant parasitic nematodes (Meyer et al. 1990; Shinya application of conidia to seed (Griffin 2007; Ownley et al. 2008). Activity of Lecanicillium spp. against et al. 2008; authors, unpublished data). both plant pathogens and insects has been demon- Endophytic B. bassiana 11-98 has been observed strated in bioassays (Askary et al. 1998; Askary and with scanning electron microscopy (SEM), and Yarmand 2007; Kim et al. 2007) and greenhouse detected with polymerase chain reaction (PCR) in studies (Kim et al. 2008) (Table 2). cotton seedlings (Griffin 2007). Using SEM on Commercial products containing Lecanicillium seedlings maintained in a sterile system, conidial spp. have not been developed for plant disease germination and hyphal growth were observed in control. However, a formulation of L. longisporum association with areas of leaf exudation. Penetration (Petch) Zare & W. Gams, known as VertalecÒ,is points through epithelial cells were observed, without available for control of insect pests. Lecanicillium formation of a specialized structure. Hyphae ramified longisporum (applied as VertalecÒ), Lecanicillium through the palisade parenchyma and mesophyll attenuatum Zare & W. Gams CS625, and Lecanicil- layers of leaf tissues. Beauveria bassiana 11-98 was lium sp. DAOM 198499 suppressed development of also detected with PCR in a mixed DNA sample of 1 powdery mildew, Podosphaera fuliginea (Schltdl.) U. part B. bassiana DNA to 1,000 parts cotton DNA, and Braun & S. Takam. (Ascomycota: Erysiphales) from surface-sterilized tissues of cotton seedlings (=synonym Sphaerotheca fuliginea) on cucumber grown from B. bassiana-treated seed (Griffin 2007; leaf discs when applied one or eight days after Ownley et al 2008; authors, unpublished data). powdery mildew inoculation. When applied to highly The results of a study with cotton seedlings infected leaf discs 11–15 days after pathogen inoc- suggested that induced systemic resistance is also a ulation, Lecanicillium treatments significantly sup- probable mechanism of biological control for pressed subsequent production of powdery mildew B. bassiana 11-98 (Griffin 2007; Ownley et al. spores, compared to controls (Kim et al. 2007). In 2008; authors, unpublished data). Isolate 11-98 was greenhouse experiments, L. longisporum (applied as evaluated for its ability to induce systemic resistance VertalecÒ) suppressed spore production of powdery in cotton against Xanthomonas axonopodis pathovar mildew on potted cucumber plants under conditions malvacearum (causes bacterial blight). Conidia of of low and high infection levels (Kim et al. 2008). B. bassiana were applied as a root drench to 5-day Askary et al. (1997) provided ultrastructural and old seedlings, 13 days prior to pathogen challenge. cytochemical evidence for the process of parasitism Treatment with B. bassiana (at 107 CFU/seedling of P. fuliginea by Lecanicillium sp. DAOM 198499 123 Endophytic fungal entomopathogens 119

Table 2 Studies on Lecanicillium spp. as dual biological controls for plant pathogens and insect pests Species or strain Type Plant pathogen Mode of action Insect Reference of Lecanicilliuma of study against plant pathogen

V. lecanii Laboratory Podosphaera fuliginea (Schltdl.) Parasitism/ Macrosiphum Askary et al. Vertalec bioassay U. Braun & S. Takam. antibiosis euphorbiae (1998) (Ascomycota: Erysiphales) (Hemiptera: DAOM 216596 (syn. Sphaerotheca Aphididae) (see below) fuliginea) Powdery mildew DAOM 198499 (see below) L. muscarium (Petch) Laboratory P. fuliginea (syn. S. fuliginea) Parasitism M. euphorbiae Askary and Zare & W. Gams bioassay Aphidius nigripes Yarmand (Ascomycota: (Hymenoptera: (2007) Hypocreales) strain Braconidae) DAOM 198499 L. longisporum (Petch) Laboratory P. fuliginea (syn. S. fuliginea) Not reported Myzus persicae Kim et al. Zare & W. Gams bioassay (Hemiptera: (2007) (Ascomycota: Aphididae) Hypocreales) M. euphorbiae (Vertalec) Aulacorthum solani L. attenuatum Zare (Hemiptera: & W. Gams Aphididae) (Ascomycota: Hypocreales) strain CS625 Lecanicillium sp. strain DAOM 198499 L. longisporum Greenhouse P. fuliginea (syn. S. fuliginea) Not reported Aphis gossypii Kim et al. (Vertalec) (Hemiptera: (2008) Aphididae) L. lecanii (Zimm.) Zare Field (survey) Hemileia vastatrix Berk. Parasitism Coccus viridis Vandermeer & W. Gams (Ascomycota: & Broome (Basidiomycota: (Hemiptera: et al. (2009) Hypocreales) Pucciniales) Coffee leaf rust Coccidae) a Name listed is the same as was given in the reference (formerly V. lecanii DAOM 198499), including has been attributed to parasitism. Indeed, an array of production of cell-wall degrading enzymes such as extracellular lytic enzymes have been reported for chitinases. They suggested that prior to invasion of P. isolates of Lecanicillium, including cellulases, prote- fuliginea, the powdery mildew fungus was weakened ases, b-1,3-glucanases, chitinases (Bidochka et al. by antibiotics produced by Lecanicillium (Askary 1999; Saksirirat and Hoppe 1991) and more recently, et al. 1997). Subsequently, Benhamou and Brodeur pectinases (Benhamou and Brodeur 2001). However, (2000) showed that this strain does produce anti- induction of plant host defense reactions against P. fungal compounds in culture that are effective against digitatum (Benhamou and Brodeur 2000; Benhamou Penicillium digitatum (Pers.) Sacc. (Ascomycota: 2004), Pythium ultimum Trow (Oomycota: Pythiales) Eurotiales), which causes postharvest green mold of (Benhamou and Brodeur 2001), and powdery mildew citrus. It has been suggested that production of (Hirano et al. 2008) have been reported. In studies on antimicrobial compounds that weaken or kill the biological control of P. ultimum, Lecanicillium sp. target host cells prior to parasitism is a form of DAOM 198499 grew intercellularly among epider- specialized saprophytism, rather than parasitism mal and cortical cells on cucumber roots treated with (Be´langer and Labbe´ 2002). the fungus (Benhamou and Brodeur 2001). Endo- In most of the studies with Lecanicillium as a phytic colonization of cucumber roots was also biological control against plant pathogens, activity observed when blastospores of L. muscarium B-2 123 120 B. H. Ownley et al. were applied to roots. Subsequently induced resis- resemble plants colonized with plant growth-promot- tance to powdery mildew on the cucumber leaf ing rhizobacteria (Harmon et al. 2004). Much of the surface was reported (Hirano et al. 2008). Koike et al. research on systemic resistance of plants infected (2004) demonstrated that L. muscarium B-2 is also a with endophytic beneficial fungi has focused on very successful epiphytic colonist of cucumber leaf mycorrhizal fungi (reviewed in Gutjahr and Pasz- surfaces, suggesting that competition for nutrients kowski 2009). These obligate fungi live on plant and space may also be operative against powdery roots and stimulate plant growth and development by mildew. increasing nutrient uptake and decreasing disease and insect problems. While plants infected with hypocrealean fungi do not have the complex structures Fungal endophytism and induced systemic associated with mycorrhizal infection, they can resistance occupy a nutritional niche in or on the plant and develop an active cross talk with their plant hosts that Recently, proteomic analysis of P. dactylifera results in induced resistance (Vinale et al. 2008). infected with endophytic B. bassiana or two Lecan- Induction of plant resistance has been reported for icillium spp. was reported by Go´mez-Vidal et al. several species of Trichoderma (Harmon et al. 2004; (2009). Colonization by B. bassiana, L. dimorphum, Jeger et al. 2009), and mechanisms for induced or L. cf. psalliotae resulted in induction of proteins resistance are beginning to emerge (Segarra et al. related to plant defense or stress response, and 2007; Vinale et al 2008). Mechanisms for induced proteins involved in energy metabolism and photo- resistance by other hypocrealean fungi are scant, but synthesis were also affected. As additional studies on much information on mechanisms of induced resis- molecular analysis of plants infected with endophytic tance obtained from studies with Trichoderma can be fungal entomopathogens are conducted, it will applied to other fungal entomopathogens. become evident that endophytism is inducing impor- Many species of Trichoderma have been commer- tant changes in plant metabolism, even though the cially developed for biological control of plant plant does not present any symptoms of endophyte diseases and insects (Harmon et al. 2004; Shakeri infection. It will be important to take into consider- and Foster 2007). Some of these isolates induce ation that endophytes may cause plants to enter a resistance to plant pathogens (Table 3). Typically, ‘‘primed state’’ (sensu Conrath et al. 2006; see also Trichoderma is applied to soil or to plant roots grown Schulz and Boyle 2005), which could be contributing in co-culture with the fungus. However, some species to the antagonistic effects of B. bassiana and induce systemic resistance when leaves are treated Lecanicillium on plant pathogenic fungi. It is also with Trichoderma conidia (Perazzolli et al. 2008; possible that endophyte infection might result in Korolev et al. 2008). Plant hosts in which resistance positive effects such as enhanced plant growth (Ernst is induced are taxonomically diverse and include both et al. 2003; Schulz and Boyle 2005). Plant growth- monocots and dicots. Several recent studies support related variables should be measured in all studies jasmonate/ethylene signaling as the mechanism for dealing with the introduction of fungal entomopath- induced systemic resistance (Table 3), further sug- ogens as possible endophytes, as was recently done gesting that the response is similar to that induced by by Tefera and Vidal (2009) for sorghum plants rhizobacteria (reviewed in Harmon et al. 2004). inoculated with B. bassiana, although it will be Induced resistance is broad spectrum, and subsequent difficult to elucidate the role of a specific endophyte challenges of the primed plant by taxonomically if others are already present in the plant. diverse pathogens (e.g., bacteria, necrotrophic fungi, When endophytism results in ‘‘primed’’ plants, biotrophic fungi) induce a rapid and intense activa- subsequent biotic challenge leads to a transitory tion of cellular defense mechanisms somewhat rem- period of strongly potentiated gene expression that is iniscent of hypersensitive responses. associated with accelerated defense responses. These Species in the genus Trichoderma (Ascomycota: responses confer broad-spectrum resistance to patho- Hypocreales) are well known for the production of gens and insects (Van Wees et al. 2008). In this bioactive metabolites that play a role in the myco- respect, plants colonized by fungal entomopathogens parasitic or entomopathogenic lifestyles of the 123 Endophytic fungal entomopathogens 121

Table 3 Recent evidence for involvement of the jasmonate/ethylene pathway in systemic resistance induced by Trichoderma species Species and strain Plant Pathogen Evidence of effects Efﬁcacy References or extract

T. asperellum Cucumis sativus Pseudomonas Significant increase of Reduced bacterial Segarra Samuels, L. (cucumber) syringae pv jasmonic acid (JA), colony forming et al. Lieckf. & Nirenberg lachrymans but not salicylic acid (SA) units by ca. 50% (2007) (Ascomycota: at 1 h, both peaked at 3 h; Hypocreales) strain JA levels not above untreated T34, (107 spores) control after 6 h, SA decreased until 24 h; Significant increase of peroxidase by 6 h T. harzianum Rifai Arabidopsis Botrytis cinerea Col-0 ecotype, and auxin- Disease severity Korolev (Ascomycota: thaliana (L.) Pers. (Ascomycota: resistant and SA acid reduced in Col-0 et al. Hypocreales) Heynh. Helotiales) mutants were ISR-inducible; following either (2008) strain T39 Mutants impaired in ABA, root or leaf gibberillic acid, or ethylene/ application JA were not ISR-inducible T. harzianum Vitis vinifera Plasmopara viticola Timing and persistence Leaf treatment Perazzolli strain T39 L. cv. Pinot (Berk. & M.A. Curtis) differed from BTH decreased et al. Noir (grape) Berl. & De Toni which is SA-dependent disease (2008) (Oomycota: severity; Root Peronosporales) treatment did not T. virens (J.H. Mill., Zea mays Colletotrichum Induction of JA and green Reduced lesion Djonovic´ Giddens & A.A. L. (corn) graminicola leaf volatile biosynthetic area in leaves et al. Foster) Arx (= Glomerella genes from endophytic (2007) (Ascomycota: graminicola plants Hypocreales) D.J. Politis strain Gv29-8 (Ascomycota: Sordariomycetidae) fungus, as well as in the induction of resistance in proteinaceous elicitor determined to be involved in plant hosts. Elicitors or resistance inducers can be induced resistance responses in rice (Oryza sativa L.), divided into three broad categories: proteins with cotton, and maize (Zea mays L.) (Djonovic´ et al. enzymatic activity, avirulence-like gene products, 2006, 2007). Recently a second small hydrophobin- and low molecular weight compounds released from like protein (Epl1) was isolated from Hypocrea cell walls (either fungal or plant) as a result of atroviride (=Hypocrea atroviridis Dodd, Lieckf. & hydrolytic enzymes (e.g., chitinase, glucanase) (Vi- Samuels (Ascomycota: Hypocreales)) (teleomorph of nale et al. 2008). In several recent studies, various T. atroviride P. Karst.) (Vargas et al. 2008). Epl1 was proteins and peptides from Trichoderma have been produced as a dimer. Sm1 can also be a dimer, but shown to induce host defense responses (Table 4). upon dimerization, the glycosyl moiety and activity Volatiles released after treatment with alamethicin, a are lost. Both hydrophobins are active as resistance 20-amino acid polypeptide isolated from T. viride inducers when configured as a monomer. Vargas Pers., affect the behavior of the parasitoid Cotesia et al. (2008) have proposed that aggregation of the glomerata (L.) (Hymenoptera: Braconidae) (Bru- elicitor disrupts the molecular cross-talk between the insma et al. 2009). Wasps chose alamethicin-treated beneficial fungal colonizer and plant. plants over nontreated plants, but chose plants on Recent proteomic studies provide a glimpse into which Pieris brassicae (L.) (Lepidoptera: Pieridae) the complexity of the Trichoderma-plant interaction. had fed over alamethicin-treated plants. In cucumber, 51 proteins were different in treatments Sm1, a hydrophobin-like small protein secreted by with T. asperellum Samuels, Lieckf. & Nirenberg and Trichoderma virens (J.H. Mill., Giddens & A.A. untreated controls; 17 proteins were up-regulated, Foster) Arx, was the first non-enzymatic and 11 were down-regulated. Proteins were divided 123 122 123 Table 4 Effects of selected Trichoderma-derived peptides and proteins on host defense responses

Peptide/protein Plant Effects and efﬁcacy Reference Similar compounds described for Beauveria or Lecanicillium spp.

Alamethicin: Ion channel- Brassica oleracea L. var. 20-fold more potent inducer of ISR than JA; Bruinsma et al. forming peptide mixture gemmifera DC. ‘Cyrus’ (brussel volatile emissions; increased preference for (2009) sprouts) parasitoid wasps (Cotesia glomerata (L.) (Hymenoptera: Braconidae)) Suspension cells of Arabidopsis Activation of callose synthase; callose Aidemark et al. thaliana (L.) Heynh. (Col-1) and deposition (2009) Nicotiana tabacum L. ‘BY-2’ (tobacco) Mitogen-activated protein kinase Phaseolus vulgaris L. (var. nanus Deletion tmk1 mutants had reduced Reithner et al. Zhang et al. (2009)—Beauveria—regulation TMK1: Serine-threonine L.) (bean) mycoparasitism and host-specific regulation (2007) of environmental stress kinases of ech42 gene transcription; deletion mutants and virulence to insects had an increased ability to protect plants against Rhizoctonia solani Ku¨hn (Basidiomycota: Cantharellales) Sm1: Cerato-platanin Zea mays L. (corn) Deletion or over-expression of Sm1 in mutants Djonovic´ et al. Ying and Feng (2004) Beauveria— protein that is hydrophobin- did not affect normal growth and development (2007) relationship between hydrophobins like of Trichoderma virens (J.H. Mill., Giddens and thermotolerance Kamp (2002) & A.A. Foster) Arx (Ascomycota: Hypocreales); Lecanicillium—Hydrophobins abundant in Root colonization was not affected in mutants, but sporulating cultures, but not in mycelial ability to induce resistance to a foliar pathogen was cultures reduced in deletion mutants and increased in some over-expression mutants Oryza sativa L. ‘M-202’ (rice); Induced expression of defense genes Djonovic´ et al. Gossypium hirsutum L. (glucanase, chitinase) locally and (2006) ‘Paymaster 2326BG/RR’ systemically; H2O2 produced in Sm1- and ‘DeltaPine 50’ (cotton) treated levels, but no resulting necrosis Ethylene-inducing xylanase: 18 Gossypium hirsutum ‘DeltaPine The 18 Kd protein increased terpenoid production Hanson and Kd protein similar to serine 50’ (cotton) and peroxidase activity Howell (2004) protease ThPG1 endopolygalacturonase: Lycopersicon esculentum ThPG1-silenced mutants had lower Morań-Diez et al. Fenice et al. (1997) Cell-wall degrading enzyme (=Solanum lycopersicon L. var. polygalacturonase activity and less growth (2009) Lecanicillium—Antarctic strains of associated with pectin lycopersicon) ‘Marmande’ on pectin medium; protection against Botrytis V.(=Lecanicillium) lecanii had wide degradation (tomato) cinerea Pers. (Ascomycota: Helotiales) was enzymatic competence, including the same for ThPG1-silenced mutants and polygalacturonase activity wild type, even though root colonization by mutants was lower al. et Ownley H. B. ABC transporter membrane L. esculentum Gene up-regulated in fungus by pathogen-secreted Ruocco et al. pump: ATP-binding cassette metabolites and some fungicides; deletion mutants (2009) with transmembrane domain were sensitive to fungicides and lost ability to protect against Pythium ultimum Trow (Oomycota: Pythiales) and R. solani Endophytic fungal entomopathogens 123 into four categories: stress and defense, energy and and reduction of surface tension to allow aerial metabolism, secondary metabolism, and protein syn- growth (Linder 2009). Hydrophobins produced thesis/folding (Segarra et al. 2007). In maize, 114 by B. bassiana have been shown to be important proteins were up-regulated and 50 were down- in conidial thermotolerance (Ying and Feng regulated in response to treatment with T. harzianum. 2004) and attachment to substrates (Holder and Most of the upregulated genes were for proteins Keyhani 2005). Hydrophobins of T. asperellum involved in carbohydrate metabolism, defense, and were proposed to protect hyphae from defense photosynthesis (Shoresh and Harman 2008). compounds during the early stages of infection There are several parallels between Trichoderma (Viterbo and Chet 2006). Therefore, it is possible and Beauveria and/or Lecanicillium spp. that suggest that they play a similar role in B. bassiana. similar mechanisms of induced resistance: Hydrophobins have been detected in Lecanicil- lium (Kamp 2002), but little is known on their 1. These fungi can live endophytically between role in the fungal life cycle. plant cells without causing negative effects on 6. Mitogen-associated protein kinases (MAP plant growth and development. Genes with sim- kinases) in the subfamily HOG-1 (High osmo- ilar function (e.g., plant defense/stress response, larity glycerol (1) are associated with host energy metabolism, and photosynthesis) are up- infection and with protection from osmotic stress regulated in plants colonized by Beauveria and in Beauveria and Trichoderma spp. The MAP Lecanicillium (Go´mez-Vidal et al. 2009) and kinases interfere with the ability of T. atroviride those colonized by Trichoderma spp. (Segarra to induce resistance to the soilborne plant et al. 2007; Shoresh and Harman 2008). pathogen, R. solani, in bean plants. Deletion 2. Plant colonization can be established horizon- mutants had a greater ability than wild type to tally by application of spores to seed, roots, or protect the plants. In B. bassiana, MAP kinases leaves. Even though the relationship between the regulated response of the fungus to stress. fungi and their hosts is intimate, plants can easily Deletion mutants were more sensitive to hyper- be infected. This is similar to mycorrhizae but osmotic stress, high temperature, and oxidative contrasts markedly with the grass endophytes in stress than the wild type (Zhang et al. 2009). the genus Neotyphodium (Ascomycota: Hypo- When transcript levels of hydrophobin-encoding creales), which are transmitted vertically via seed genes in the deletion mutants were low, conidial (Gimeńez et al. 2007; Hartley and Gange 2009). attachment to cicada hind wings was severely 3. Beauveria and Trichoderma spp. are natural and impaired (Zhang et al. 2009). introduced colonists of a wide variety of plants 7. Both Beauveria and Trichoderma spp. can that include both dicots and monocots. Although induce systemic resistance to bacterial patho- there is less information available on the plant gens. In cucumber, plants infected by T. asper- host range of Lecanicillium spp., it has also been ellum (107 conidia ml-1) supported less than recovered as a natural and introduced endophyte 50% the number of colony-forming units (CFU) of monocots and dicots. after challenge with Pseudomonas syringae 4. All three fungi produce a wide array of enzymes pathovar lachrymans (Segarra et al. 2007). and avirulence-like products. Hydrolytic Treatment of cotton with 1 9 107 CFU B. enzymes that can attack substrates as diverse as bassiana 11-98 per root induced systemic resis- plant cell walls, insect cuticle, and oomycetous tance against bacterial blight (Xanthomonas and fungal plant pathogens are important for the axonopodis pathovar malvacearum) on cotton varied nutritional niches occupied by these fungi. foliage. Although bacterial populations were not 5. Beauveria bassiana and many species of Trich- assessed, foliar disease ratings were significantly oderma produce hydrophobins or hydrophobin- lower for Beauveria-treated plants than the like molecules. It has been suggested that the untreated control (Griffin 2007). functions of hydrophobins in the life cycle of 8. Both Lecanicillium and Trichoderma spp. can fungi include: formation of protective layers, induce systemic resistance to oomycetous plant attachment, structural components of cell walls, pathogens. Host plant signaling and subsequent 123 124 B. H. Ownley et al.

intense defense responses have been proposed for fungicide resistance in these pathogens (Fry 1982). Lecanicillium-treated cucumber. Ingress of P. The ability of the hypocrealean fungi to use several ultimum into roots resulted in the deposition of strategies reduces the probability of development of an electron-opaque material that frequently encir- resistance. For example, treatment of roots or seeds cled pathogen hyphae and accumulated in unin- with Beauveria or Lecanicillium spp. conidia poten- fected xylem vessels (Benhamou and Brodeur tially produces endophyte-infected plants that reduce 2001). Inoculation of roots with L. muscarium initial establishment of the disease through induced resulted in root colonization and endophytic resistance. Studies have shown that both Beauveria growth. Plant leaves were protected from powdery and Lecanicillium spp. can become established as mildew, but defense enzymes were not different in epiphytes, which provides opportunities for plant colonized and non-colonized plants (Hirano et al. disease suppression through antibiosis, competition, 2008). Trichoderma harzianum induced systemic or mycoparasitism. Endophytic and epiphytic popu- resistance in pepper plants grown from seed lations of these fungi could also reduce insect damage treated with T. harzianum spores (Ahmed et al. to the plant. 2000). Stem lesions, caused by inoculation with Plant diseases caused by soilborne fungi are Phytophthora capsici Leonian (Oomycota: Per- notoriously difﬁcult to control since these fungi onosporales), were 40% shorter than lesions in generally have wide host ranges and can survive in inoculated plants grown from non-treated seed. P. soil for long periods of time as saprophytes or as capsici was isolated from zones immediately specialized survival structures (e.g., sclerotia, chlam- contiguous with the necrotic tissue, but T. harzia- ydospores). Resistant cultivars are available for a num was not, suggesting that there was no direct limited number of host-pathogen combinations. Soil- contact between them. The percentage of P. borne pathogens often cause disease at multiple life capsici isolated nine days after inoculation was stages of the plant (i.e., seed rot, damping-off of greater in non-treated inoculated plants than in seedlings, and root rots), but typically, the greatest Trichoderma-treated plants inoculated with P. impact is on the seed or newly emerged seedling. Use capsici. In addition to induced resistance against of hypocrealean fungi as plant, seed, or soil treat- P. capsici in the upper part of the plant, concen- ments facilitates rapid colonization of plant hosts and tration of the phytoalexin capsidiol was more than creates potential for subsequent induced resistance. 7-fold greater than in non-treated plants inocu- Older plants may be protected from root rots by lated with P. capsici, six days after inoculation induced systemic resistance, although this has not (Ahmed et al. 2000). been documented. Seed treatment may also create a potential ‘antibiotic’ spermosphere that inhibits pop- Conclusions ulations of seed rot pathogens. Mycoparasitism by hypocrealean fungi can be directed against survival The ability of many hypocrealean entomopathogens structures of soilborne plant pathogens, thus reducing to occupy nutritional niches as diverse as insects, their inoculum potential. fungi, and plants provides unique opportunities for Although much has been accomplished in the biological control of multiple plant pathogens and commercial development of Beauveria and Lecani- insect pests. Use of these fungi may overcome some cillium spp. as fungal entomopathogens in plant of the challenges faced in plant disease control. For production, more work is needed to understand the example, many foliar phytopathogens have a very roles of these fungi as epiphytes and endophytes high sporulation rate and are well-suited for wide- involved in suppression of plant diseases. Some spread dissemination as air-borne propagules. If strains of these fungi have been approved for use as genetic resistance is not available in the crop, bioinsecticides. Use in plant disease control extends fungicide applications are often the primary means development of these products. Future studies should of disease control. The rapid reproduction rate of focus on the ecology of these fungi (Vega et al. foliar pathogens coupled with frequent applications 2009a, b), their role in plant-microbe interactions, of systemic fungicides, many of which are narrow and their antagonism against pathogenic and nontar- spectrum, increases the chances of developing get microorganisms. 123 Endophytic fungal entomopathogens 125

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