Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10¹⁰ bacterial cells ml⁻¹, a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10⁹ and 10⁸ cells ml⁻¹. Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT₅₀ = 17.1 h) than for Steinernema carpocapsae (RT₅₀ = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect’s food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.     

芒果、香蕉采后病害生物防治的研究进展

芒果、香蕉采后主要病害为炭疽病、蒂腐病、冠腐病、黑腐病、黑星病.生物防治是当前芒果、香蕉采后病害控制的重要研究方向.概述了生物防治芒果、香蕉采后病害的方法,包括诱抗剂、植物提取物、拮抗微生物在芒果、香蕉采后病害防治上的研究与应用.     

Quantification of Pseudomonas fluorescens strains F113, CHA0 and Pf153 in the rhizosphere of maize by strain-specific real-time PCR unaffected by the variability of DNA extraction efficiency

Pseudomonas fluorescens strains F113 and CHA0 are well-known plant growth-promoting rhizobacteria (PGPR) often used as model strains in biocontrol experiments. To monitor their persistence in large scale field experiments, culture-independent methods are needed. In this study, a strain-specific real-time PCR quantification tool was developed based on sequence-characterized amplified regions (SCAR) for P. fluorescens strains F113, CHA0 and Pf153. Differences in DNA extraction efficiencies from rhizosphere samples were circumvented using plasmid APA9 as internal standard to normalize C_T values after real-time amplification. The detection limits of the real-time PCR assays for all three strains were approximately 10 cells for genomic DNA and 10⁴ cells/g rhizosphere for maize samples grown in different natural soils. Population sizes of the three strains in the rhizosphere of maize measured by the new real-time PCR approaches were similar to those measured by most probable number (MPN)-PCR. A persistence study of the three strains indicated that the strains persisted differently over a period of 5 weeks. In conclusion the newly developed real-time PCR approach is a fast and resource efficient method for monitoring individual biocontrol strains in natural soil, which makes it an apt quantification tool for future large-scale field experiments.     

Drying of Epicoccum nigrum conidia for obtaining a shelf-stable biological product against brown rot disease

AIMS: The effects of freeze-drying, spray-drying and fluidized bed-drying on survival of Epicoccum nigrum conidia were compared. METHODS AND RESULTS: Viability of E. nigrum conidia (estimated by measuring its germination) was 100% after fluidized bed-drying and freeze-drying, but it was determined that skimmed milk must be added in the case of freeze-drying conidia. Addition of other protectants (Tween-20, peptone, sucrose, glucose, starch and peptone + starch) to skimmed milk before freeze-drying did not improve the conidial viability which was obtained with skimmed milk alone. Glycerol had a negative effect on the lyophilization of E. nigrum conidia. Epicoccum nigrum conidia freeze-dried with skimmed milk, or fluidized bed-dried alone maintained an initial viability for 30 and 90 days, respectively, for storage at room temperature. Epicoccum nigrum conidial viability after spray-drying was lower than 10%. CONCLUSIONS: The best method to dry E. nigrum conidia was fluidized bed-drying. Conidia without protectants dried by this method had 100% viability and survived for 90 days at room temperature. SIGNIFICANCE AND IMPACT OF STUDY: This paper deals with methods for the potential formulation of a biocontrol agent which is being tested for eventual commercialization.     

Chitinolytic enzymes: their contribution to basic and applied research

After cellulose, chitin is the second most abundant renewable resource available in nature. Marine invertebrates and fungal biomass are the two main sources of chitinous waste, which is commercially exploited. The enzymes involved in chitin degradation have been particularly well studied. Such enzymes have applications in ultrastructural studies, in the preparation of chitooligosaccharides which show anti-tumour activity, as biocontrol agents and in single-cell protein production. Here, the contribution chitin enzymology can make to basic and applied research is discussed.     

Fermentation and toxin studies of the molluscicidal strains ofBacillus brevis

Summary Strain SS86-4 was one of 40Bacillus brevis strains shown to be molluscicidal to the schistosomiasis snail vectorBiomphalaria glabrata. When grown in mB4 medium in 2-L fermentors, SS86-4 was molluscicidal only if fructose or phenylalanine was present in the medium. This is reminiscent of secondary fermentation factor effects, in this case an antioxidant effect. In vivo proteases also were capable of reducing molluscicidal activity. The molluscicidal toxin has an LC₅₀ of 1 g toxin protein ml^–1 (approx. 1 p.p.m.) and may be described as a small proteinaceous, heat-stable, oxygen-sensitive entity associated with the particulate portion of the cell wall fraction ofB. brevis that is formed prior to sporulation. Initial information indicates that its HPLC signature shows major peaks at 148.37 and 163.96 s and consists of two bands of approximately 5.3 kDa and 8.7 kDa on PAGE gel.     

Purification and evaluation of 2, 4-di-tert butylphenol (DTBP) as a biocontrol agent against phyto-pathogenic fungi

A fungal strain, Marasmiellus sp (PUK64), isolated from the mangrove forests in Muthupet, Tamil Nadu, East coast of India, along with others were screened for the search of potent bioactive compounds. A phenolic compound, 2,4-di-tert-butylphenol (DTBP), was isolated from the most promising strain PUK64 and its chemical structure was ascertained. DTBP demonstrated remarkable antifungal activity against the phytopathogenic fungi Aspergillus oryzae, Curvularia lunata and Fusarium verticillioides. In an in-vitro experimental setup, DTBP suppressed the growth of all three fungi, among which F. verticillioides was found to be highly susceptible. This effect relates with the inhibition of spore germination and hyphal growth that we observed. DTBP showed high affinity with the F. verticillioides's β-tubulin protein (determined by ligand-protein docking) as compared to the standard fungicide carbendazim (CBZ). Molecular docking and simulation studies of DTBP with target β-tubulin further confirmed the potential of β-tubulin binding in F. verticillioides. To our knowledge, this is the first report on DTBP-mediated biocontrol of phytopathogenic fungi, produced by Marasmiellus sp. PUK64 that can be potent inhibitor of β-tubulin protein of F. verticillioides.     

Moderate drought influences the effect of arbuscular mycorrhizal fungi as biocontrol agents against Verticillium-induced wilt in pepper

Previous studies have shown that the arbuscular mycorrhizal fungus (AMF) Glomus deserticola (Trappe, Bloss and Menge) can diminish the negative effect of Verticillium dahliae Kleb. on pepper yield. On the other hand, it is known that AMF can be more beneficial for plant growth and physiology under dry conditions than when soil moisture is plentiful. Therefore, our objective was to assess if a moderate water deficit imposed on pepper plants before their inoculation with V. dahliae could improve the effectiveness of G. deserticola as biocontrol agent. In the present experiment, the delay in disease development in Verticillium-inoculated plants associated with AMF did not occur under well watered conditions. In addition, the establishment of mycorrhizal symbiosis and the development of structures by AMF were delayed when both symbiotic and pathogenic fungi infected the same root. Therefore, it is suggested that the equilibrium between pepper plant, G. deserticola and V. dahliae is so complex that small changes in competition between symbiotic and pathogenic fungi for host resources can modify the efficiency of AMF as a biocontrol agent. On the other hand, water deficit enhanced the deleterious effect of V. dahliae on fruit set and yield only when pepper plants were not associated with G. deserticola, which reinforces the idea that AMF may be more important for host plants subjected to stressful conditions. However, comparing well watered non-mycorrhizal and predroughted mycorrhizal plants, we found that moderate water deficit imposed before inoculation with V. dahliae did not improve the effectiveness of G. deserticola as a biocontrol agent.     

Accumulation of Compatible Solutes in Penicillium frequentans Grown at Reduced Water Activity and Biocontrol of Monilinia laxa

The biocontrol fungus Penicillium frequentans was grown on potato dextrose broth of either - 0.4 MPa water potential (unstressed) or reduced water potential (stressed) media modified with glycerol to - 7.0 MPa. The endogenous compatible solutes and sugars were quantified after 5, 10 and 15 days growth. Glycerol was the main compatible solute accumulated at reduced water potential, with a concomitant decrease in erythritol and mannitol, and the sugar, glucose. Maximum accumulation of glycerol occurred in 5-day-old cultures, with the content in P. frequentans biomass produced at reduced water potential being about 180 times higher than that produced in unmodified medium (- 0.4 MPa). There was no enhanced accumulation of the desiccation protectant trehalose. Homogenisation of cultures, for spray applications of the fungus, resulted in a partial loss of the solutes accumulated in the fungal biomass. P. frequentans controlled peach brown rot ( Monilinia laxa ) on peach twigs, with the disease index being significantly smaller when treated with the antagonist produced at reduced water potential. However, both inoculum types were able to reduce the length of lesions to a similar extent. This suggests that the inoculum of the antagonist produced in medium with freely available water (- 0.4 MPa) becomes rapidly adapted to the dry environment in the phyllosphere, counteracting the possible initial advantage of the inoculum produced at reduced water potential (- 7.0 MPa).