Fungal Root Endophytes from <i>Musa</i> spp. as Biological Control Agents against the Plant Pathogen <i>Fusarium oxysporum</i>

Freshwater fungus Anguillospora longissima was recovered as root endophytic fungus from the pteridophytic plant Equisetum sp., growing in riparian areas of Nainital, Kumaun Himalaya, and evaluated through well diffusion and dual culture methods. The isolated endophytic fungus was found a potent inhibitor for test pathogenic fungi. Maximum inhibition by the isolated endophytic fungus was showed against pathogenic fungus Tilletia indica (79.76%), while minimum was recorded against Fusarium oxysporum (35.73%), in dual culture method. Further, in well diffusion method, pathogenic fungi Colletotrichum falcatum and Pyricularia oryzae were found maximum (62.96%) and minimum (17.85%) inhibited fungi, respectively, by the isolated fungus. In the present investigation, the used root endophytic fungus A. longissima was found potent to produce some antifungal substances that inhibited the growth of all test pathogenic fungi.

Many root fungal endophytes inhabiting forest trees have potential impact on the health and disease progression of certain tree species. Hence, the screening of root endophytes for their biocontrol abilities is relevant for their potential to protect their hosts against invaders. The aim of this research is to screen for the potential inhibitory effects of selected conifer root endophytes during interaction, in vitro, with the root rot pathogen, Heterobasidion parviporum. Here, we introduce a guideline that facilitates the use of root fungal endophytes as biocontrol agents. We isolated fungal root endophytes from eight different conifers. These root fungal endophytes were evaluated for their antagonism against the root rot pathogen, H. parviporum, by means of paired-culture antagonism assays. We determined the antagonism of the isolated root fungal endophytes to elucidate potential biocontrol applications. For the analysis, a software package in R was developed. Endophyte candidates with antagonistic potential were identified.

Fusarium wilt caused by Fusarium oxysporum f. sp. nicotianae (Johnson) W. C. Snyder and H. N. Hansen. is the most destructive disease of tobacco. The present study was carried out in vitro conditions to assess the possible use of biocontrol agents in field conditions. Trichoderma viride, Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens were evaluated for their antagonistic activity against F. oxysporum f. sp. nicotianae under in vitro conditions. Different biocontrol agents showed varying degrees of antagonism. T. viride and T. harzianum showed 70 and 40% inhibition of radial growth of the F. oxysporum f. sp. nicotianae respectively in dual culture technique. Mode of parasitism between the biocontrol agents and F. oxysporum f. sp. nicotianae were studied under the microscope. Formation of several loops and coiling of hyphae by T. viride and T. harzianum around the hyphae of the pathogen forming a thick compact rope like structure was observed. The biocontrol agents produced volatile metabolites. These metabolites suppressed the growth of F. oxysporum f. sp. nicotianae. The two biocontrol agents varied in the extent of volatile metabolite production. Among bacteria, B. subtilis inhibited the radial growth of F. oxysporum f. sp. nicotianae by 36%. The bacterial and fungal antagonists were not mutually antagonistic as the co-inoculation did not inhibit each other. The present work is the first report on the use of T. viride, T. harzianum and B. subtilis against F. oxysporum f. sp. nicotianae as potential biocontrol agents.

Fusarium wilt caused by Fusarium oxysporum f. sp. nicotianae (Johnson) W. C. Snyder and H. N. Hansen. is the most destructive disease of tobacco. The present study was carried out in vitro conditions to assess the possible use of biocontrol agents in field conditions. Trichoderma viride, Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens were evaluated for their antagonistic activity against F. oxysporum f. sp. nicotianae under in vitro conditions. Different biocontrol agents showed varying degrees of antagonism. T. viride and T. harzianum showed 70 and 40% inhibition of radial growth of the F. oxysporum f. sp. nicotianae respectively in dual culture technique. Mode of parasitism between the biocontrol agents and F. oxysporum f. sp. nicotianae were studied under the microscope. Formation of several loops and coiling of hyphae by T. viride and T. harzianum around the hyphae of the pathogen forming a thick compact rope like structure was observed. The biocontrol agents produced volatile metabolites. These metabolites suppressed the growth of F. oxysporum f. sp. nicotianae. The two biocontrol agents varied in the extent of volatile metabolite production. Among bacteria, B. subtilis inhibited the radial growth of F. oxysporum f. sp. nicotianae by 36%. The bacterial and fungal antagonists were not mutually antagonistic as the co-inoculation did not inhibit each other. The present work is the first report on the use of T. viride, T. harzianum and B. subtilis against F. oxysporum f. sp. nicotianae as potential biocontrol agents.

Medicinal plants harbor tremendously diverse bacterial endophytes that maintain plant growth and health. In the present study, a total of 124 culturable bacterial endophytes were isolated from healthy Aconitum carmichaelii Debx. plants. These strains were clustered into 10 genera based on full-length 16S rDNA sequences, among which Bacillus and Pseudomonas were the dominant genera. In addition, A. carmichaelii may capture 10 potential new bacterial species based on multi-locus sequence analysis of three housekeeping genes (gyrA, rpoB, and atpD). The majority of these bacterial endophytes exhibited plant growth-promoting ability through diverse actions including the production of either indole acetic acid and siderophore or hydrolytic enzymes (glucanase, cellulose, and protease) and solubilization of phosphate or potassium. A total of 20 strains inhibited hyphal growth of fungal pathogens Sclerotium rolfsii and Fusarium oxysporum in vitro on root slices of A. carmichaelii by the dual-culture method, among which Pseudomonas sp. SWUSTb-19 showed the best antagonistic activity. Field experiment confirmed that Pseudomonas sp. SWUSTb-19 significantly reduced the occurrence of southern blight and promoted plant biomass compared with non-inoculation treatment. The possible mode of actions for Pseudomonas sp. SWUSTb-19 to antagonize against S. rolfsii involved the production of glucanase, siderophore, lipopeptides, and antimicrobial volatile compounds. Altogether, this study revealed that A. carmichaelii harbored diverse plant growth-promoting bacterial endophytes, and Pseudomonas sp. SWUSTb-19 could be served as a potential biocontrol agent against southern blight.

The use of living beneficial microbes and/ or their products to control plant pathogens can offer a safer alternative to chemical treatments. In this study, needle-leaf fungal endophytes (NLE) were isolated from symptoms-free needle-leaves of two host plants collected in Cavite and Batangas, Philippines. A total of 73 NLEs were observed from three tree samples for each of the angiosperm Casuarina equisetifolia Engl. and the gymnosperm Pinus kesiya Royle ex Gordon. These were identified as belonging to 17 morphospecies. Of these, seven NLEs, mainly isolated from C. equisetifolia, were tested for their antagonistic activities against three potential plant pathogens, Fusarium oxysporum s.l. Smith & Swingle, F. solani s.l. (Mart.) Sacc., and F. moniliforme s.l. J. Sheld. using the dual-culture method at three strategies. Our results showed NLEs inhibited F. oxysporum on contact via the preventive, eradicative, and simultaneous approaches indicating that fungal endophytes may be used as potential biocontrol agents against F. oxysporum s.l..

Evaluation of Pseudomonas and Bacillus Strains as Potential Biocontrol Agents against Fusarium Wilt of Chickpea

Screening of antagonistic yeast strains for postharvest control of Penicillium expansum causing blue mold decay in table grape

Surveys (in 2002 and 2003) were performed for fungal endophytes in roots of 24 plant species growing at 12 sites (coastal and inland soils, both sandy soils and salt marshes) under either water or salt stress in the Alicante province (Southeast Spain). All plant species examined were colonized by endophytic fungi. A total of 1830 fungal isolates were obtained and identified by morphological and molecular [internal transcribed spacer (ITS) and translation elongation factor-1alpha gene region (TEF-1alpha) sequencing] techniques. One hundred and forty-two fungal species were identified, belonging to 57 genera. Sterile mycelia were assigned to 177 morphospecies. Fusarium and Phoma species were the most frequent genera, followed by Aspergillus, Alternaria and Acremonium. Fungal root endophytic communities were influenced by the soil type where their respective host plants grew, but not by location (coastal or inland sites). Fusarium oxysporum, Aspergillus fumigatus and Alternaria chlamydospora contributed most to the differences found between endophytic communities from sandy and saline soils. Host preference was found for three Fusarium species studied. Fusarium oxysporum and Fusarium solani were especially isolated from plants of the family Leguminosae, while Fusarium equiseti showed a preference for Lygeum spartum (Gramineae). In some cases, specificity could be related to intra-specific variability as shown by sequencing of the TEF-1alpha in the genus Fusarium.

Antagonistic activity of bacteria and fungi from horticultural compost against Fusarium oxysporum f. sp. melonis

ABSTRACTThe current study aimed to identify the survival of bio-control bacteria with antifungal activity against Fusarium oxysporum and assess their growth promoting activity in wheat crop field conditions. To evaluate the fungicidal activities of isolated bacteria using the dual culture method, both qualitative and quantitative bioassays were performed. Plant Growth Promoting activities such as Indole 3-Acetic Acid (IAA), phosphate solubilization, Hydrogen cyanide (HCN), and Siderophore production were assessed for three biocontrol bacterial isolates (BCB 07, BCB16, and BCB 83) out of 180 with 70% antagonistic activity against Fusarium oxysporum. Chitinase, protease, and cellulase interaction in isolates was also tested. BCB16 was selected as it had 70% antagonist activity against F. oxysporum but also had the highest PGPR (Plant Growth Promoting Rhizobacteria) traits when compared to the other two isolates. BCB16 was also tested for survival in talc powder and in wheat crop field conditions. Even after 4 months in talc powder, the survival rate remained stable. In a wheat crop field, BCB16 reduced the disease incidence of Fusarium oxysporum by 54.38%. When compared to fungus alone treatment, BCB16 increased average yield by 57% alone and 32% in challenged conditions. BCB16 was identified molecularly using the 16s rRNA gene. Bacillus amyloliquefaciens shared 97% of the deduced sequence. The sequence was submitted to genbank and assigned the accession number OM333889. Bacillus amyloliquefaciens has the potential to be used in the field as an alternative to synthetic fungicides against Fusarium oxysporum.

In nature, production of disease-free plants with enhanced yield and compounds of therapeutic value can be mediated through rhizospheric microorganisms. There are increasing environmental concerns over the widespread use of biocontrol measures in general, and alternatively, more sustainable methods of disease control are now being sought. Plant diseases caused by root pathogens need to be controlled in order to maintain the quality and abundance of food, feed and fiber, the prime necessities of life. Different approaches are used for prevention and control of these root pathogens. Among these alternatives are those referred to as biological control; the most obvious and apparently biological control is a potent means of reducing the damage caused by plant pathogens. The potential agents for biocontrol activity are rhizosphere-competent fungi and bacteria which, in addition to their antagonistic activity, are capable of inducing growth responses by either controlling minor pathogens or by producing growth-stimulating factors. A variety of biological controls are available for use, but further development and effective adoption requires a greater understanding of the complex interactions among plants, people, and the environment. This article emphasizes: (1) information about mycorrhiza and root endophytes, (2) various definitions and key mechanisms of biocontrol, and (3) the relationships between microbial diversity and biological control.

Plants are affected by various biotic and abiotic stresses due to climate change. Tomato and carrots are important crops that are attacked by various pathogens. Fourteen plant-growth-promoting bacteria (PGPB) belonging to the genera Streptomyces sp. and Nocardiopsis sp. were selected for the biocontrol of several common fungal and bacterial pathogens. Antifungal activity was assessed against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) and Rhizoctonia solani (RHS). Antibacterial activity was evaluated against Pseudomonas syringae (PS), Pseudomonas corrugata (PC), Pseudomonas syringae pv. actinidiae (PSA), and Pectobacterium carotovorum subsp. Carotovorum (PCC). In vitro antifungal and antibacterial antagonistic activities were evaluated by the dual culture method. Fungal–bacterial interaction areas were analyzed by scanning electron microscopy (SEM). Cell-free culture filtrates (CFs) from strains showing good biocontrol potential were produced and investigated for their in vitro antifungal and antibacterial activity. The two most effective strains were also combined in consortium and utilized for In Planta pre-emergence biocontrol assays on both S. lycopersicum and D. carota. For each pathogenic strain, four experimental conditions were compared: CNT (no PGPB/no infection), PGPB (with PGPB/no infection), PGPB+INF (with PGPB/with infection), and INF (no PGPB/with infection). Streptomyces albidoflavus strain H12 and Nocardiopsis aegyptica strain H14 showed good in vitro antifungal (inhibition of >50%) and antibacterial (inhibition halo of >10 mm) activity. The SEM micrographs showed deterioration of fungal filaments and modification of hyphal structures. The CFs of both strains were also able to inhibit FORL and RHS in in vitro growth (minimum inhibitory concentration of 0.2–0.8%). In planta biocontrol assessments showed that the consortium was effective in reducing the infection effects of both fungal and bacterial pathogens. Dual consortium allowed regular plant development compared to the control. These results confirm the usefulness of actinomycetes strains as a biocontrol agent and can therefore be an alternative to chemicals used in agriculture.

Next generation biological control – an introduction

Biocontrol strategies have been mainly focused on proposing the use of biocontrol agents (BCAs) isolated from the rhizospheric region of the plant for protection against phytopathogens. In the present study six isolates of Trichoderma sp. were isolated and identified. Among them, two isolates were identified as Trichoderma harzianum, two as T. asperellum, one as T. virens, and the rest one identified as T. hamatum. These isolates were identified molecularly by using Internal Transcribed Spacer (ITS) primers amplification by polymerase chain reaction and submitted to National Center for Biotechnology Information (NCBI). All identified isolates along with isolate ITCC-5593 were screened for their antagonistic activity using dual culture method. Trichoderma harzianum isolates (ThL-1, ThL-4 and 5593) were found more potent antagonists against Fusarium oxysporum f. sp. lentis (Fol) NFLRJ-3. Selected isolates of Trichoderma sp. after dual culture assay were further studied for the production of cell wall-degrading enzymes (chitinase and β-1,3 glucanase) against NFLRJ-3 along with production of phyto-hormones i.e. Indole Acetic Acid (IAA) and Gibberllic Acid (GA3) two isolates namely ThL-4 and 5593 gave maximum production of hormones. The elicitation of the defense related enzymes in the plants were analysed using biochemical assays for important enzymes, i.e. Peroxidase (PO), Polyphenol oxidase (PPO) and Super oxide dismutes (SOD) level in challenged plants over untreated and unchallenged control plants. It was found that T. harzianum secretes two major cell wall-degrading enzymes, i.e., chitinase and β-1,3 glucanase. Optimum production of both the enzymes were found at 96 h of incubation and maximum activity was showed by isolate 5593 i.e. 9.16 and 2.86 U/mg protein, respectively. The role of T. harzianum isolates in inducing defense enzymes (PO, PPO and SOD) in lentil when exposed to Fol was also observed. It was found that T. harzianum (ThL-4 and 5595) induced higher levels of enzymes. This study proposes the use of rhizospheric Trichoderma sp. isolates for better and all around protection of plants against soil and seed borne phytopathogens.