Involvement of lytic enzymes and secondary metabolites produced by Trichoderma spp. in the biological control of Pythium myriotylum.

This investigation was aimed to evaluate the antimicrobial activities and involvement of extracellular lytic enzymes produced by four strains of Trichoderma in the inhibition of Pythium myriotylum. Antagonistic effects were tested by dual culture. Activities of lytic enzymes were evaluated from the filtrate of each strain after cultivation in selected media. Organic extracts were obtained from liquid media subsequent to the cultivation of Trichoderma in potato dextrose broth (PDB). Non-volatile organic compounds such as polyphenols and flavonoids were evaluated spectrophotometrically while volatile organic compounds (VOCs) were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). The antimicrobial activity of the organic extracts was determined using the poisoning method. Results have shown that all the strains were antagonists against P. myriotylum. T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11) that were found to produce cellulase, protease, and xylanase. Over 20 compounds were identified in each extract, including esters, lactones, and organic acids. The organic extracts also contained high amounts of polyphenolic compounds and flavonoids and significantly inhibited the mycelial growth of P. myriotylum. The minimal inhibition concentrations were 80 µg/µL, 40 µg/µL, 20 µg/µL, and 10 µg/µL, for extracts obtained from T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11), respectively. There was significant correlation between the production of total polyphenol and flavonoid content and the antagonistic effects of the tested strains.

Morphology, Ultrastructure, and Molecular Phylogeny of Rozella multimorpha, a New Species in Cryptomycota.

Increasing numbers of sequences of basal fungi from environmental DNA studies are being deposited in public databases. Many of these sequences remain unclassified below the phylum level because sequence information from identified species is sparse. Lack of basic biological knowledge due to a dearth of identified species is extreme in Cryptomycota, a new phylum widespread in the environment and phylogenetically basal within the fungal lineage. Consequently, we are attempting to fill gaps in the knowledge of Rozella, the best-known genus in this lineage. Rozella is a genus of unwalled, holocarpic, endobiotic parasites of hosts including Chytridiomycota, Blastocladiomycota, Oomycota, Basidiomycota, and a green alga, with most species descriptions based on morphology and host specificity. We found a Rozella parasitizing a Pythium host that was a saprobe on spruce pollen bait placed with an aquatic sample. We characterized the parasite with light microscopy, TEM of its zoospores and sporangia, and its 18S/28S rDNA. Comparison with other Rozella species indicates that the new isolate differs morphologically, ultrastructurally, and genetically from Rozella species for which we have data. Features of the zoospore also differ from those of previously studied species. Herein we describe the Rozella as a new species, R. multimorpha.

Biological Control of Diseases of Vegetables Grown Hydroponically in Thailand: Challenge and Opportunity.

BACKGROUND: In Thailand, yield loss due to plant diseases in vegetables grown hydroponically is very high as a result of the growers` lack of knowledge for controlling diseases and their un- willingness to invest in setting-up the proper hydroponic system from the beginning. Severe root rot disease caused by Pythium spp. is frequent and can be anticipated in the hot climate in Thailand. OBJECTIVE: This review focuses on the diseases in temperate lettuces which have been produced hydroponically and have been attacked by plant pathogens, particularly Pythium spp. METHODS: Biological control of vegetable diseases grown hydroponically has been investigated in Thailand. Research is being carried out to identify effective strains of the antagonists, formulating the applicable products and delivering them appropriately to control the disease. Products of Bacillus subtilis, Chaetomium globosom and Trichoderma harzianum have been recommended for use to control diseases in vegetables grown hydroponically. RESULTS: Control efficacy of these products is varied as the biological products have been used by the growers in the paradigm of using chemical fungicide for disease control in hydroponic production system, overlooking the intrinsic characteristics of the biological control products. CONCLUSION: The recent patent, which minimizes the effects of sunlight and heat on the nutrient solution without the use of an external energy for cooling the nutrient, should be applied in producing hydroponic vegetables to mitigate poor plant growth and root rot disease outbreak in Thailand.

Genetic, physiological and biochemical characterization of Bacillus sp. strain RMB7 exhibiting plant growth promoting and broad spectrum antifungal activities.

BACKGROUND: Plant growth promoting rhizobacteria (PGPR) are functionally diverse group of bacteria having immense potential as biofertilizers and biopesticides. Depending upon their function, they may serve as partial replacements for chemical fertilizer or pesticides as an eco-friendly and cost-effective alternatives as compared to their synthetic counterparts. Therefore, isolation, characterization and practical evaluation of PGPRs having the aforementioned multifaceted beneficial characteristics, are essentially required. This study describes the detailed polyphasic characterization of Bacillus sp. strain RMB7 having profound broad spectrum antifungal activity and plant growth promoting potential. RESULTS: Based on 16S rRNA gene sequencing, strain RMB7 was identified as Bacillus specie. This strain exhibited the production of 8 mg. L(-1)of indole-3-acetic acid (IAA) in tryptophan-supplemented medium. It was able to solubilize 50.6 mg. L(-1) tri-calcium phosphate, reduced 601ηmol acetylene h(-1)/vial and inhibited >70% growth of nine fungal phytopathogens tested in vitro. Under natural pathogen pressure, inoculation with strain RMB7 and RMB7-supernatant conferred resistance by arugula plant against Pythium irregulare with a concurrent growth improvement over non-inoculated plants. The T-RFLP analysis based on 16S rRNA gene showed that inoculation with RMB7 or its supernatant have a major impact on the indigenous rhizosphere bacterial population. Mass spectrometric analysis revealed the production of lipopeptide surfactins as well as iturin A presence in crude extract of RMB7. PCR-amplification further confirmed the presence of genes involved in the biosynthesis of these two bioactive lipopeptide compounds. CONCLUSIONS: The data show that Bacillus sp. strain RMB7 has multifaceted beneficial characteristics. It may be an ideal plant growth promoting as well as biocontrol agent, for its integrated use in disease and nutrient management strategies.

Biological control of plant pathogens: advantages and limitations seen through the case study of Pythium oligandrum.

The management of certain plant beneficial microorganisms [biological control agents (BCAs)] seems to be a promising and environmental friendly method to control plant pathogens. However, applications are still limited because of the lack of consistency of BCAs when they are applied in the field. In the present paper, the advantages and limitations of BCAs are seen through the example of Pythium oligandrum, an oomycete that has received much attention in the last decade. The biological control exerted by P. oligandrum is the result of a complex process, which includes direct effects through the control of pathogens and/or indirect effects mediated by P. oligandrum, i.e. induction of resistance and growth promotion. P. oligandrum antagonism is a multifaceted and target fungus-dependent process. Interestingly, it does not seem to disrupt microflora biodiversity on the roots. P. oligandrum has an atypical relationship with the plant because it rapidly penetrates into the root tissues but it cannot stay alive in planta. After root colonisation, because of the elicitation by P. oligandrum of the plant-defence system, plants are protected from a range of pathogens. The management of BCAs, here P. oligandrum, is discussed with regard to its interactions with the incredibly complex agrosystems.

Influence of Pythium oligandrum on the bacterial communities that colonize the nutrient solutions and the rhizosphere of tomato plants.

The influence exerted by the biocontrol oomycete Pythium oligandrum on the bacterial populations proliferating in the rhizosphere of tomato plants grown in a hydroponic system and in the circulating solutions is studied in the present experiment. Quantitative PCR and single-strand conformation polymorphism were used to investigate the genetic structure and dynamics of the bacterial communities colonizing the root systems and the various circulating solutions. Quantitative PCR assays showed that bacteria heavily colonized the rhizosphere of tomato plants with, however, no significant density changes throughout the cultural season (April-September). Single strand conformation polymorphism fingerprints revealed the occurrence of transient perturbations in the rhizospheric indigenous bacterial communities following P. oligandrum introduction in the root system of plants. This effect was, however, transient and did not persist until the end of the cropping season. Interestingly, the genetic structure of the bacterial microflora colonizing either the roots or the nutrient solutions evolved throughout the cropping season. This temporal evolution occurred whatever the presence and persistence of P. oligandrum in the rhizosphere. Evidence is also provided that bacterial microflora that colonize the root system are different from the ones colonizing the circulating solutions. The relationships between these 2 microflora (at the root and solution levels) are discussed.

Ecology of root colonizing Massilia (Oxalobacteraceae).

BACKGROUND: Ecologically meaningful classification of bacterial populations is essential for understanding the structure and function of bacterial communities. As in soils, the ecological strategy of the majority of root-colonizing bacteria is mostly unknown. Among those are Massilia (Oxalobacteraceae), a major group of rhizosphere and root colonizing bacteria of many plant species. METHODOLOGY/PRINCIPAL FINDINGS: The ecology of Massilia was explored in cucumber root and seed, and compared to that of Agrobacterium population, using culture-independent tools, including DNA-based pyrosequencing, fluorescence in situ hybridization and quantitative real-time PCR. Seed- and root-colonizing Massilia were primarily affiliated with other members of the genus described in soil and rhizosphere. Massilia colonized and proliferated on the seed coat, radicle, roots, and also on hyphae of phytopathogenic Pythium aphanidermatum infecting seeds. High variation in Massilia abundance was found in relation to plant developmental stage, along with sensitivity to plant growth medium modification (amendment with organic matter) and potential competitors. Massilia absolute abundance and relative abundance (dominance) were positively related, and peaked (up to 85%) at early stages of succession of the root microbiome. In comparison, variation in abundance of Agrobacterium was moderate and their dominance increased at later stages of succession. CONCLUSIONS: In accordance with contemporary models for microbial ecology classification, copiotrophic and competition-sensitive root colonization by Massilia is suggested. These bacteria exploit, in a transient way, a window of opportunity within the succession of communities within this niche.

Plant growth promotion and biological control of Pythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes.

AIMS: To evaluate the potential of Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis endophytic in cucumber roots, to promote plant growth and to protect seedlings and mature plants of cucumber from diseases caused by Pythium aphanidermatum, under greenhouse conditions. METHODS AND RESULTS: Three endophytic isolates, out of 29, were selected through tests aimed at understanding their mechanisms of action as biocontrol agents and plant growth promoters. When applied individually or in combination, they significantly promoted plant growth and reduced damping-off and crown and root rot of cucumber. The combination of the three isolates resulted in significantly better suppression of diseases and plant growth promotion, than where the plants were exposed to individual strains. CONCLUSIONS: The three selected actinomycete isolates colonized cucumber roots endophytically for 8 weeks, promoted plant growth and suppressed pathogenic activities of P. aphanidermatum on seedling and mature cucumber plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The results clearly show that the endophytic, glucanase-producing actinomycetes used, especially as a combined treatment, could replace metalaxyl, which is the currently recommended fungicide for Pythium diseases in the United Arab Emirates. These endophytic isolates also have the potential to perform as plant growth promoters, which is a useful attribute for crop production in nutrient impoverished soils.

Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism against Pythium ultimum.

The involvement of the G-alpha protein GNA3 in the production of cell wall-degrading enzymes (CWDEs) by Trichoderma reesei during antagonism against Pythium ultimum was investigated. cAMP content was 2.8-fold higher in the T. reesei mutant gna3QL than in the parental TU-6. The gna3QL, like TU-6, inhibited the growth of P. ultimum in dual culture assays. Scanning electron microscopy showed that the gna3QL promoted more morphological alterations of P. ultimum cell wall than TU-6. In general, gna3QL produced higher activities of CWDEs than TU-6. We therefore suggest that CWDEs production during mycoparasitism by T. reesei against P. ultimum may be associated with the level of GNA3 activity.

Rhizosphere Pseudomonas sp. strains reduce occurrence of pre- and post-emergence damping-off in chile and tomato in Central Himalayan region.

Based on in vitro screening for PGP and anti-mycelial activity against three zoosporic pathogenic oomycetes, Pythium aphanidermatum 123, P. aphanidermatum 4746, and Phytophthora nicotianae 4747, seven bacterial isolates were selected for field trials on tomato and chile to test for plant growth promotion under natural and artificial disease-infested field sites in both winter and wet seasons. The effectiveness of isolates in the field trials correlated with the in vitro antagonism screening data. Pseudomonas sp. FQP PB-3, FQA PB-3 and GRP(3) showed substantial beneficial effects on plant growth promotion and lowered considerably the incidence of pre- and post-emergence damping-off in both the crops under various disease scenarios. For example, seed bacterization with these bacterial strains reduced pre-emergence-damping off by ca. 60-70% in the two natural sites, with and without histories of fungicide use in the winter season, and to a lesser extent, ca. 20-40%, in the warmer wet (high humidity; 85-92%) season. The suppression efficacy for post-emergence damping-off was less compared to pre-emergence damping-off although still significant (P > 0.05). Our data unambiguously show that screening of a large number of bacterial pool identifies promising isolates that show beneficial effects on all stages of plant growth in natural oomycete-infested regimes.

Inability to find consistent bacterial biocontrol agents of Pythium aphanidermatum in cucumber using screens based on ecophysiological traits.

A collection of 821 rhizobacteria from cucumber, originating from different root locations and stages of plant development, was screened for potential biocontrol agents of Pythium aphanidermatum (Edson) Fitzp. The screening procedure exploited carbon source utilization profiles and growth rates of bacteria as indicators of a partial niche overlap with the pathogen. The bacteria were tested for growth on nine carbon sources (glucose, fucose, sucrose, maltose, asparagine, alanine, galacturonic acid, succinic acid, and linoleic acid), most of which are reported to be used by the zoospores of P. aphanidermatum in the infection process. The isolates were classified as fast- or slow-growing, depending on their growth rate in 1/10 strength TSB. By nonhierarchical cluster analysis, 20 clusters were generated of bacteria with similar profiles of carbon source utilization. Redundancy analysis showed that the type of root sample explained 47% of the variance found in the relative abundance of bacteria from the clusters. Bacteria from clusters using none or few of the carbon sources, e.g., maltose and linoleic acid, with many slow-growing isolates, showed a preference for plants in the vegetative or generative stage, or for old root regions (root base). Bacteria from clusters with fast-growing isolates, using many carbon sources, were relatively abundant in the seedling stage. A selection of 127 bacteria from the different clusters was tested for disease suppressive capabilities in bioassays on young cucumber plants in nutrient solution, inoculated with zoospores of P. aphanidermatum. Nine of these bacteria produced biosurfactants, and 27 showed antibiosis against mycelial growth in plate assays. For 31 isolates, significant positive effects on plant biomass were shown, as analyzed with a general linear regression model. For most isolates, these effects occurred only in one of two replicate assays and no reductions in the degree of root and crown rot were found. Of the isolates that used many of the tested carbon sources, only four had positive effects on plant biomass. The majority of the isolates that positively affected plant biomass used few to moderate numbers of carbon sources and did not produce antibiotics or biosurfactants. In conclusion, competition for the tested carbon sources with the zoospores did not play a decisive role in disease suppression, and no clear relation was found between ecophysiological traits and disease suppression. Only isolate 3.1T8, isolated from root tips in the generative stage of plant growth, significantly increased plant biomass and suppressed root and crown rot symptoms in five out of six bioassays. The isolate produced an antifungal substance in plate assays and showed biosurfactant production in several (cucumber-derived) media.

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists.

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.

An integrated control of Pythium root rot of greenhouse tomato.

Pythium root rot caused by Pythium aphanidermatum is one of the most important diseases of greenhouse tomatoes. Hydroponic culture exacerbates the problem. Both nutrient film technique (NFT) and recirculating growing systems pose a challenge in the control of this disease, because the pathogen, especially the zoospores, can spread easily in the recirculating solution to the whole growing system. Fortunately, hydroponically grown plants are easier to manipulate than soil grown plants, proper manipulation of root environments can lead to excellent disease control. This paper reports the development of an effective integrated control measure for pythium root rot of tomato by integrating pH, bioagent, and ultra-violet irradiation in a specific manner. This integrated control consists of three operations: a) before transplanting, the UV system is connected to sterilize the recirculating solution using 100 mJcm-2; b) after transplanting, the nutrient solution is delivered at pH 5.0 regime for five weeks followed by adjusting pH to 5.8 to 6.2 regime for one week; and c) bacterial bioagent, such as Pseudomonas is introduced into the root zone at 100 mL per plant at 10(8) bacteria mL-1 or added to the nutrient solution to arrive at 10(6) bacteria mL-1 in the solution. This report also discusses the advantages and limitations of this measure in the control of pythium root rot.

Pythium invasion of plant-based life support systems: biological control and sources.

Invasion of plant-based life support systems by plant pathogens could cause plant disease and disruption of life support capability. Root rot caused by the fungus, Pythium, was observed during tests of prototype plant growth systems containing wheat at the Kennedy Space Center (KSC). We conducted experiments to determine if the presence of complex microbial communities in the plant root zone (rhizosphere) resisted invasion by the Pythium species isolated from the wheat root. Rhizosphere inocula of different complexity (as assayed by community-level physiological profile: CLPP) were developed using a dilution/extinction approach, followed by growth in hydroponic rhizosphere. Pythium growth on wheat roots and concomitant decreases in plant growth were inversely related to the complexity of the inocula during 20-day experiments in static hydroponic systems. Pythium was found on the seeds of several different wheat cultivars used in controlled environmental studies, but it is unclear if the seed-borne fungal strain(s) were identical to the pathogenic strain recovered from the KSC studies. Attempts to control pathogens and their effects in hydroponic life support systems should include early inoculation with complex microbial communities, which is consistent with ecological theory.

Genetic basis in plants for interactions with disease-suppressive bacteria.

Plant health depends, in part, on associations with disease-suppressive microflora, but little is known about the role of plant genes in establishing such associations. Identifying such genes will contribute to understanding the basis for plant health in natural communities and to new strategies to reduce dependence on pesticides in agriculture. To assess the role of the plant host in disease suppression, we used a genetic mapping population of tomato to evaluate the efficacy of the biocontrol agent Bacillus cereus against the seed pathogen Pythium torulosum. We detected significant phenotypic variation among recombinant inbred lines that comprise the mapping population for resistance to P. torulosum, disease suppression by B. cereus, and growth of B. cereus on the seed. Genetic analysis revealed that three quantitative trait loci (QTL) associated with disease suppression by B. cereus explained 38% of the phenotypic variation among the recombinant inbred lines. In two cases, QTL for disease suppression by B. cereus map to the same locations as QTL for other traits, suggesting that the host effect on biocontrol is mediated by different mechanisms. The discovery of a genetic basis in the host for interactions with a biocontrol agent suggests new opportunities to exploit natural genetic variation in host species to enhance our understanding of beneficial plant-microbe interactions and develop ecologically sound strategies for disease control in agriculture.