Endophytic Establishment Research Articles

Endophytic establishment of native Bacillus thuringiensis strain in maize plants and its efficacy against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

BackgroundBacillus thuringiensis (Bt) is known as the most successful microbial insecticide worldwide used against lepidopteran insect pests in agriculture. Native Bacillus isolate VKK5 showing insecticidal activity against Spodoptera frugiperda (FAW) (J.E. Smith) (Lepidoptera: Noctuidae) was characterized as B. thuringiensis (BtVKK5) on a morphological and molecular basis. Recent research has shown that Bt can be established as an endophytic organism for controlling insect pests. The present work aimed at assessing the colonization of BtVKK5 as an endophyte in five maize cultivars by seed treatment (ST), soil drenching (SD), foliar application (FA) and combination of all methods (ST + SD + FA) and its bioefficacy against neonates of FAW.ResultsEstablishment of inoculated BtVKK5 as endophytes in five maize cultivars, viz. Pusa HQPM7 Improved, Pusa Jawahar Hybrid Maize 1, Pusa Vivek Hybrid 27 Improved (PVH27I), Pusa HQPM5 Improved and DMRH 1301, was confirmed by re-isolating from the leaves of the plant on ampicillin-selected agar plates. Estimation of colony-forming units per gram of leaf showed that there was a significant difference in colonization of the Bt strain among maize cultivars by different inoculation methods. The colonies were further substantiated by the amplification of cry1A and cry1E genes. Bioefficacy studies showed the highest mortality (50%) in the ST + FA + SD inoculation method, followed by ST (40%) in PVH27I. Moreover, growth inhibition was observed in survived larvae on inoculated plants vis-a-vis control.ConclusionEstablishment of Bt strain as an endophyte in maize plants, complemented with insecticidal activity, could possibly lead to an innovative approach to the management of S. frugiperda and other borers.

Temporal Dynamics of Endogenous Bacterial Composition in Rice Seeds During Maturation and Storage, and Spatial Dynamics of the Bacteria During Seedling Growth.

Seed endophytes are of interest because they are believed to affect seed quality, and ultimately, plant growth and fitness. A comprehensive understanding of the assembly of the seed microbiome during seed development and maturation, the fate of microbes during storage, and the migration of microbes during seedling growth are still lacking. In this study, to understand the assembly and fate of endogenous bacteria in rice seeds from the ripening stage to the storage and seedling stages, we employed culture-dependent and metagenomic analyses. Bacterial communities in rice seeds were composed of a few dominant taxa that were introduced at the milky and dough stages, and they persisted during seed maturation. The culturable bacterial population gradually increased during the ripening stage, whereas there was a gradual decrease during storage. Bacteria that persisted during storage proliferated after imbibition and were distributed and established in the shoots and roots of rice seedlings. The storage temperature influenced the abundance of bacteria, which consequently changed the bacterial composition in the shoots and roots of seedlings. Pantoea, Pseudomonas, and Allorhizobium were consistently abundant from seed development to the germination stage. Some endogenous bacterial strains significantly promoted the growth of Arabidopsis and rice plants. Overall, our results indicate that rice seeds are colonized by a few bacterial taxa during seed development, and their relative abundance fluctuates during storage and contributes significantly to the establishment of endophytes in the stems and roots of rice plants. The selected bacterial isolates can be used to improve the growth and health of rice plants. To the best of our knowledge, this is the first study to reveal the dynamics of bacterial populations during storage of rice seeds at different temperatures. The temporal dynamics of the bacterial community during seed storage provide clues for the manipulation of endogenous bacteria in rice plants.

Effect of Plant Systemic Resistance Elicited by Biological and Chemical Inducers on the Colonization of the Lettuce and Basil Leaf Apoplast by Salmonella enterica.

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S. Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H2O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S. Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H2O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S. Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and dl-β-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H2O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1, as did Salmonella apoplastic colonization in H2O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

Interactions Between Two Invertebrate Pathogens: An Endophytic Fungus and an Externally Applied Bacterium.

The members of family Noctuidae exist in diverse environments and many species from this group are of agriculture importance, particularly Helicoverpa spp. Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is a major pest of many legumes and cereal crops. Due to environmental and regulatory concerns, safe alternatives to broad spectrum chemical insecticides are needed for the control of key noctuid pests such as H. armigera. A strain of Beauveria bassiana (Cordycipitaceae: Hypocreales) was evaluated for its ability to colonize endophytically in chickpea plants, and its effectiveness against second (L2) and fourth (L4) larval instars of H. armigera. B. bassiana was inoculated to chickpea plants through injection and endophytic establishment was confirmed by re-isolating the fungi from leaf samples. A detached leaf assay was used to evaluate pathogenicity. Bacillus thuringiensis was also applied to both larval stages through leaf dip method. In a novel approach, combined treatments of bacteria and endophytic fungi were compared with single-pathogen treatments. Relative to the single treatments, the combined pathogen treatments exhibited an increase in larval mortality, and decrease in pupation, adult emergence and egg eclosion. Specifically, synergistic effects on mortality were observed when larvae were exposed to simultaneous application of B. bassiana (1 × 108 conidia ml–1) with B. thuringiensis (0.75 μg ml−1). Both instars exhibited varying level of growth, development, frass production, diet consumption and fecundity when exposed to the chickpea leaves inoculated with endophytic B. bassiana and dipped with sub-lethal doses of B. thuringiensis. These findings indicate that the integrated application of endophytic colonized B. bassiana and B. thuringiensis can be effectively used against H. armigera.

Endophytic establishment of the fungal entomopathogen, Beauveria bassiana (Bals.) Vuil., in cucumber plants

BackgroundThe fungus, Beauveria bassiana (Bals.) Vuil., is one of the most important entomopathogenic fungi (EPFs). Recently, its new role was discovered in nature, to be an endophyte in plants. It has been reported as an endophytic fungus in many monocotyledonous and dicotyledonous plants.Main bodyThe study was conducted to evaluate the ability of the fungus, B. bassiana, to colonize and persist in cucumber plants under laboratory conditions and to detect its systemic growth inside the plant tissues in addition to pathogenicity in the plant. The isolate, B195, of the fungus, B. bassiana, was used. Five different inoculation methods were followed: seed dusting, seed immersion, soil drench, seedling drench, and foliar spray. The fungus, B. bassiana, could persist inside different cucumber tissues up to 90 days from inoculation. Soil drench provided the highest recovery rates, while foliar spray gave the lowest rates. Colonization rates reached 94.44 and 73.68% for stem and 68.26 and 37.79% for root, 30 and 90 days post soil drench, respectively, while in foliar spray, it reached 33.51 and 16.45%, after 30 and 90 days post-treatment, for the stem and 9.45 and 0% for the root, respectively. No negative effects were observed in inoculated plants or on fungal pathogenicity.ConclusionResults showed for the first time the ability of the fungus, B. bassiana, isolate B195, to artificially colonize and survive in different parts of cucumber plants under laboratory conditions by different inoculation methods and to grow systemically in plant tissues. This study is considered a preliminary study to the utilization of the fungus, B. bassiana, as an endophyte in cucumber plants to reduce the density of insect pests.

Screening, identification, and colonization of fungal root endophytes against Dematophora necatrix: a ubiquitous pathogen of fruit trees

The goal of the present research was to ascertain the potential root endophytic fungi against Dematophora necatrix, Hartig, the causal pathogen of white root rot in apples; however, it has an expanded range of hosts across different fruit trees. Out of 36 endophytic fungi segregated from symptomless roots of apple, wheat, maize, marigold, cherry, and garlic plants, only 9 isolates showed inhibitory effect during preliminary screening and were further assayed under in vitro, pot, and field conditions against the white root rot pathogen/disease. Under in vitro conditions, maximum mycelial inhibition of 81.48% was obtained with the isolate Aspergillus aculeatus strain C2. Microscopic studies on interaction between fungal endophyte with hyphal tips of D. necatrix revealed various morphological abnormalities in the hyphae. In glasshouse conditions, seed treatment pursued by soil application with Crinipellis tabtim strain M8 isolate was highly effective and exhibited 93.55% disease control. Similarly, under field conditions, the overall maximum disease control was exhibited by Crinipellis tabtim strain M8 (84.95%). The most promising root endophytes that were identified rely on morphological and ITS sequence analysis. Root colonization assay was performed which revealed maximum endosphere and rhizosphere colonization with Crinipellis tabtim strain M8. Additionally, confocal microscopic illustrations of transverse sections of root cells tenanted by fungal endophytes as compared to untreated control suggested the persistence and establishment of endophytes in the endosphere of apple seedlings. These findings present the first report on colonization of apple roots by fungal root endophytes suggesting an alternative and sustainable approach for management of white root rot disease.

Endophytic establishment of Beauveria bassiana and Metarhizium anisopliae in maize plants and its effect against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) larvae

The aim of the present study was to evaluate the artificial establishment of Beauveria bassiana and Metarhizium anisopliae as endophytes in maize plants, and its effect in controlling the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) larvae. The commercial strains B. bassiana Bb-18 and M. anisopliae Ma-30 were concentrated at 1 × 108 conidia ml−1, and the soil drench method was used to establish them as endophytes in maize plant parts. The biological control assays were conducted under laboratory conditions on second and fourth larval instars of S. frugiperda. B. bassiana colonized roots, stems, and leaves of maize tissues. However, a high occurrence of B. bassiana was obtained in roots than leaves and stems with 25, 10, and 5 isolations, respectively, whereas M. anisopliae was only acquired on roots. Both entomopathogenic fungi caused (100%) mortality on the second instar larvae. In addition, B. bassiana and M. anisopliae killed (87 and 75%) of the fourth larval instars, respectively. The fungus M. anisopliae caused the highest sporulation rates during the study. These results suggest that the entomopathogenic fungi might contribute to a sustainable S. frugiperda management in maize production in Cuba.

Endophytic Beauveria bassiana activates expression of defence genes in grapevine and prevents infections by grapevine downy mildew Plasmopara viticola

Fungal entomopathogens like Beauveria bassiana (Ascomycota: Hypocreales) are known as antagonists of insects with multiple functional and ecological roles, and have attracted increased attention as biocontrol agents in integrated pest management programmes. For some crop plants, it has been proven that endophytic B. bassiana, besides its entomopathogenic habit, can provide protection against plant pathogens or limit their damaging effects. However, for grapevine, limited knowledge is available on the influence of endophytic B. bassiana on fungal pathogens and about the mechanisms underlying putative protection effects. This study assessed the protective potential of endophytic B. bassiana against grapevine downy mildew Plasmopara viticola in greenhouse experiments. Three and seven days after a B. bassiana treatment, potted grapevine plants were inoculated with P. viticola and the evolving disease severity was assessed. Disease severity was significantly reduced in B. bassiana‐treated plants compared to control plants, depending on the age of leaves. Furthermore, a microarray and an RT‐qPCR analysis were performed to work out fundamental aspects of genes involved in the interaction between grapevine and B. bassiana. The results indicate an up‐regulation of diverse defence‐related genes in grapevine as a response to endophytic establishment of B. bassiana. Thus, endophytic establishment of an entomopathogenic fungus such as B. bassiana in grapevine plants would represent an alternative and sustainable plant protection strategy, with the potential for reducing pesticide applications in viticulture.

Endophytic Colonization and Plant Growth Promoting Effect by Entomopathogenic fungus, Beauveria bassiana to Red Chili (Capsicum annuum L.) with Different Inoculation Methods

Beauveria bassiana (Bals.) Vuill is one of entomopathogenic fungus, that be able to endophytically colonize different plants. Beauveria bassiana can promote the growth of red chili following their endophytic establishment within plants through seed treatment, soil drenching and foliar spraying. The aims of this study were: (i) To get the best colonisation B. bassiana from different isolates and to differentiate inoculation methods, i.e. seed immersion, soil drenching and foliar spraying, for the endophytic inoculation of B. bassiana in red chili; and (ii) to assess the effect of B.bassiana to stimulate or trigger plant growth from isolate Wheat, Coffea, Cacao and Leptocorisa acuta. Our result showed that all of B. bassiana isolate were able to colonize seedling of chili and B.bassiana of wheat isolate was the best isolate giving spuring seedling or plant growth. From the three inoculation methodes, inoculation B. bassiana by foliar spraying was the highest colonisation. The highest colonization was in leaves, and the second were in stem. Root plant was the poorly colonized by B. bassiana fungus.

Endophytic Beauveria bassiana in maize affects survival and fecundity of the aphid Sitobion avenae

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.

Seed inoculation with endophytic fungal entomopathogens promotes plant growth and reduces crown and root rot (CRR) caused by Fusarium culmorum in wheat.

Fungal entomopathogens, Beauveria bassiana (NATURALIS) and Metarhizium brunneum (BIPESCO5), can promote the growth of wheat following their endophytic establishment within plants through seed treatment. Similar to endophytic B. bassiana which has already been reported as a disease antagonist by several previous studies, the present study demonstrates that M. brunneum can suppress disease pathogens following plant colonization as well. 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.

Fungal entomopathogens as endophytes reduce several species of Fusarium causing crown and root rot in sweet pepper (Capsicum annuum L.)

Fusarium Crown and Root Rot (FCRR) is one of the most widespread and economically important diseases caused by a complex of Fusarium spp. infecting a wide crop range worldwide. Although there are many studies on the efficacy of bacterial and fungal biocontrol agents against FCRR, there have been no attempts to investigate the efficacy of entomopathogenic fungi as endophytes for FCRR biocontrol. At the same time, the antagonistic activity of endophytic fungal entomopathogens against plant pathogens remains understudied compared to their well-known biocontrol activity against insect pests. Therefore, the present study was initiated to examine for the first time the antagonistic activity of the fungal entomopathogens Beauveria bassiana (strain NATURALIS) and Metarhizium brunneum (strain BIPESCO5) against three Fusarium species (F. oxysporum, F. culmorum, and F. moniliforme) causing FCRR in vitro through dual-culture tests and in planta following the endophytic establishment of fungal entomopathogens in pot-culture experiments. Both fungal entomopathogens significantly inhibited all Fusarium species in vitro, with competition for niche or resources and antibiosis as modes of action. Further in planta bioassays showed that plants endophytically colonized with fungal entomopathogens had significantly reduced FCRR incidence and severity as well as improved growth, irrespective of fungal entomopathogen or Fusarium spp. This study demonstrates for the first time the biocontrol potential of endophytic fungal entomopathogens against several Fusarium species causing FCRR. It is also the first report for the suppressive activity of M. brunneum as an endophyte against plant pathogens.

Probiotics for Plants? Growth Promotion by the Entomopathogenic Fungus Beauveria bassiana Depends on Nutrient Availability.

Cultivation of crops requires nutrient supplements which are costly and impact the environment. Furthermore, global demands for increased crop production call for sustainable solutions to increase yield and utilize resources such as nutrients more effectively. Some entomopathogenic fungi are able to promote plant growth, but studies over such effects have been conducted under optimal conditions where nutrients are abundantly available. We studied the effects of Beauveria bassiana (strain GHA) seed treatment on the growth of maize (Zea mays) at high and low nutrient conditions during 6weeks in greenhouse. As expected, B. bassiana seed treatment increased plant growth, but only at high nutrient conditions. In contrast, the seed treatment did not benefit plant growth at low nutrient conditions where the fungus potentially constituted a sink and tended to reduce plant growth. The occurrence of endophytic B. bassiana in experimental plant tissues was evaluated by PCR after 6weeks, but B. bassiana was not documented in any of the above-ground plant tissues indicating that the fungus-plant interaction was independent of endophytic establishment. Our results suggest that B. bassiana seed treatment could be used as a growth promoter of maize when nutrients are abundantly available, while the fungus does not provide any growth benefits when nutrients are scarce.

Maize seed endophytic bacteria: dominance of antagonistic, lytic enzyme-producing Bacillus spp.

Beneficial aspects of endophytic microorganisms have motivated researchers to explore plant endophytic world. The present study was aimed to isolate and characterize the seed-borne endophytic bacteria from diverse maize genotypes.Eighty maize seed endophytic bacteria (MSEB), isolated from 30 maize genotypes, were characterized using polyphasic approach. The dendrograms and phylogenetic tree generated on the basis of ARDRA analysis and metabolic profiling of endophytic bacteria revealed genotypic and biochemical diversity among MSEB. The 16S rDNA sequence analysis revealed Bacillus as the most dominant encountered genus affiliated with Phylum Firmicutes. Few isolates belonged to genus Staphylococcus, whereas one isolate was identified as Corynebacterium sp. under Phylum Actinobacteria. Majority of the MSEB isolates exhibited antagonism against phytopathogenic fungi, production of ammonia, and secretion of lytic enzymes; some isolates also exhibited indole acetic acid production, the traits of which can be helpful in endophytic establishment and advantageous to the host plant. Besides, many MSEB exhibited tolerance to salinity (10%), osmotic stress (40% PEG6000), and temperature (60°C), indicating their possible application under stress conditions. Endophytic nature of the selected MSEB isolates was confirmed by tracking their presence in shoots, leaves, and roots of the host seedlings with the help of biochemical marker (rifampicin resistance). Thus, the MSEB identified in the present study can be explored as potential bioinputs for improving plant growth and productivity under stressed conditions, besides helping in understanding the plant-endophyte interactions.

Encapsulation of Metarhizium brunneum enhances endophytism in tomato plants

Biocontrol of insect pests using fungal entomopathogens has been promoted as a promising alternative to chemical pesticides. Apart from their direct mode of action against insect pests, several fungal entomopathogens have been reported as natural endophytes, paving the way for novel plant protection measures. In this study, we aimed at the selective propagation of finely dispersed Metarhizium brunneum strain BIPESCO5 mycelium in submerged culture and encapsulation in calcium alginate/starch beads to protect the fungus during drying, enable growth on different soils and promote endophytism in tomato plants. We found that a combination of osmotic pressure and increased medium viscosity promoted selective formation of finely dispersed mycelium reflected by 34.1-fold decreased pellet diameters, 1.8-fold increased mycelial biomass concentrations and low blastospore contents of 40.4×105mL−1 after 48h. Encapsulation of mycelium enhanced drying survival by 31.5%. Co-encapsulated starch was degraded by 24.8% after 21days and served as a nutrient source for growth on soils with best results on sterile and non-sterile potting substrate with 8.7mm and 4.4mm radial mycelial growth, respectively, compared to water agar with 7.6mm. When applied to the soil, encapsulation significantly increased endophytism 3.8–7.0-fold compared to plants treated with non-formulated fungal biomass. This study provides the first evidence of endophytic establishment of M. brunneum in the stem of tomato plants after application of mycelium to roots as well as on increased endophytism by encapsulation. These results might provide the basis for future work on increasing endophytism by formulation technologies.

Global expression analysis of the response to microaerobiosis reveals an important cue for endophytic establishment of Azoarcus sp. BH72.

The endophyte Azoarcus sp. BH72, fixing nitrogen microaerobically, encounters low O2 tensions in flooded roots. Therefore, its transcriptome upon shift to microaerobiosis was analyzed using oligonucleotide microarrays. A total of 8.7% of the protein-coding genes were significantly modulated. Aerobic conditions induced expression of genes involved in oxidative stress protection, while under microaerobiosis, 233 genes were upregulated, encoding hypothetical proteins, transcriptional regulators, and proteins involved in energy metabolism, among them a cbb3 -type terminal oxidase contributing to but not essential for N2 fixation. A newly established sensitive transcriptional reporter system using tdTomato allowed to visualize even relatively low bacterial gene expression in association with roots. Beyond metabolic changes, low oxygen concentrations seemed to prime transcription for plant colonization: Several genes known to be required for endophytic rice interaction were induced, and novel bacterial colonization factors were identified, such as azo1653. The cargo of the type V autotransporter Azo1653 had similarities to the attachment factor pertactin. Although for short term swarming-dependent colonization, it conferred a competitive disadvantage, it contributed to endophytic long-term establishment inside roots. Proteins sharing such opposing roles in the colonization process appear to occur more generally, as we demonstrated a very similar phenotype for another attachment protein, Azo1684. This suggests distinct cellular strategies for endophyte establishment.

Endophytic Beauveria bassiana in grapevine Vitis vinifera (L.) reduces infestation with piercing-sucking insects

Fungi are important natural pathogens of arthropod pests and are successfully used as biocontrol agents in various crops. In addition to colonizing arthropods, evidence has accumulated that some entomopathogenic fungi like Beauveria bassiana can endophytically colonize a wide array of plant species. However, only limited information is currently available on the endophytic colonization of grapevines with B. bassiana and whether the fungus still maintains its pathogenic habit against insect pests.Greenhouse and field experiments were conducted to optimize endophytic establishment of the entomopathogenic fungus B. bassiana in younger, potted plants and mature grapevine plants in the vineyard. We used two different commercialized B. bassiana strains, applied either as conidial suspensions (ATCC 74040 and GHA) or as a formulated product (Naturalis®, strain ATCC 74040) on grapevine leaves. The potential of endophytic B. bassiana to provide protection against putative target pest insects like the vine mealybug Planococcus ficus was assessed in a bioassay using surface sterilized leaves. Endophytic survival of B. bassiana inside leaf tissues of seven-week-old potted plants was evident for at least 21days after inoculation, irrespective of the inoculum used. Endophytic B. bassiana reduces infestation rate and growth of P. ficus. In the vineyard B. bassiana was detected as an endophyte in mature grapevine plants up to five weeks after last application with significant reduction of infestation with grape leafhopper, Empoasca vitis.

Root environment is a key determinant of fungal entomopathogen endophytism following seed treatment in the common bean, Phaseolus vulgaris

The common bean is the most important food legume in the world. We examined the potential of the fungal entomopathogens Beauveria bassiana and Metarhizium anisopliae applied as seed treatments for their endophytic establishment in the common bean. Endophytic colonization in sterile sand:peat averaged ca. 40% higher for fungus treatments and ca. six times higher for volunteer fungi (other fungal endophytes naturally occurring in our samples), relative to sterile vermiculite. Colonization by B. bassiana and M. anisopliae was least variable in sterile vermiculite and most variable in sterile soil:sand:peat. The impact of soil sterilization on endophytic colonization was assessed in a separate experiment using six different field-collected soils. Soil sterilization was the variable with the largest impact on colonization (70.8% of its total variance), while the fungal isolate used to inoculate seeds explained 8.4% of the variance. Under natural microbial soil conditions experienced by common bean farmers, seed inoculations with B. bassiana and M. anisopliae are unlikely to yield predictable levels of endophytic colonization.

Wheat seed embryo excision enables the creation of axenic seedlings and Koch's postulates testing of putative bacterial endophytes.

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Endophytic Ability of Different Isolates of Entomopathogenic Fungi Beauveria bassiana (Balsamo) Vuillemin in Stem and Leaf Tissues of Maize (Zea mays L.).

The present study was conducted to examine the ability of six promising indigenous isolates of Beauveria bassiana (NBAII-Bb-5a, 7, 14, 19, 23 and 45) as an endophyte in maize stem and leaf tissues. Maize seedlings (var. Nithyashree) were inoculated with conidial suspensions and were examined for endophytic establishment in leaf and stems at different intervals during 15-90days after treatment. All six isolates showed colonization in stem and leaf tissues with varying abilities of colonization and persistence. The mean percent colonization ranged from 7.41 to 20.37% in older stem tissues and 3.70 to 21.29% in young stem tissues and in leaf, it ranged from 6.46 to 27.78% in older leaf tissues and 11.11 to 26.85% in young leaf tissues. Among six isolates tested, Bb-23 isolate recorded the maximum mean colonization in older stem (20.37%), older leaf (27.78%) and in young stem (21.29%). Bb-5a isolate showed maximum mean colonization in young leaf tissues (26.85%). Persistence of inoculated fungal isolates decreased with increase in age of the plant. No physical symptoms of damage were observed in any of the B. bassiana treated plants. No colonization of B. bassiana was observed in the untreated control maize plants. The results obtained in plating and PCR techniques were similar with regard to the confirmation of endophytic establishment of B. bassiana. This study indicated the possibility of using B. bassiana as an endophyte in maize for management of maize stem borer, Chilo partellus.