Isolation,evaluation and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> from cotton rhizospheric soil with biocontrol activity against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Present investigation is based on the isolation of Bacillus subtilis from cotton rhizosphere and their evaluation as biocontrol agent against Fusarium oxysporum. The production of extracellular hydrolytic enzyme was studied for determining the antagonism. 43% of 21 isolates were identified under the B. subtilis group on the basis of biochemical characterization. 38% isolates showed competitive activity against Fusarium oxysporum and exhibit more than 50% mycelial inhibition in dual culture bioassay. The pot assay of cotton by seed treatment and soil amendment technique under green house condition showed the competent activity of the isolates in preventing the wilting of cotton seedlings due to F. oxysporum infection. SVI values of 30 day old seedlings indicated that the soil inoculation with B. subtilis BP-2 and seed treatment with B. subtilis BP-9 significantly promoted the growth of cotton seedlings. RAPD profiling revealed the diversity in the Bacillus subtilis group, ranging from 10 to 32%. The discriminative pattern among the isolates belonging to the same species was validated by 16S rDNA partial sequencing which identified them into four different strains of B. subtilis.     

Long-term orchard groundcover management systems affect soil microbial communities and apple replant disease severity

Apple replant disease (ARD) is a soil-disease syndrome of complex etiology that affects apple tree roots in replanted orchards, resulting in stunted tree growth and reduced yields. To investigate whether different groundcover management systems (GMSs) influence subsequent ARD severity, we grew apple seedlings in an outdoor nursery in pots containing orchard soil from field plots where four GMSs had been maintained for 14 years in an orchard near Ithaca, NY, USA. The GMS treatments were: (1) pre-emergence herbicide (Pre-H), bare soil strips maintained by applying tank-mixed glyphosate, norflurazon and diuron herbicides annually; (2) post-emergence herbicide (Post-H), sparse weed cover maintained by applying glyphosate in May and July each year; (3) mowed sod grass (Mowed Sod); and (4) bark mulch (Mulch). Soils were also sampled from the grass drive lane maintained between the trees in the orchard (Grass Lane). Sampled soils (Orchard soil) were either pasteurized or left untreated, placed into 4-L pots, and planted with one apple seedling per pot. After 3 months of growth, soil (Bioassay soil) and apple tree roots (Bioassay roots) were sampled from each pot and microbial populations colonizing samples were characterized. Seedling growth was reduced in soils sampled from all four GMS treatments compared to the Grass Lane soils. Among the GMS treatments, seedling biomass was greater in Pre-H than in the Post-H soil. Soil microbial communities and nutrient availability differed among all four GMS treatments and the Grass Lane. Root-lesion (Pratylenchus sp.) nematode populations were higher in the Mowed Sod than in the other GMS treatments. Soil bacterial and fungal community composition was assessed in Orchard and Bioassay soils and Bioassay roots with a DNA fingerprinting method (T-RFLP). Redundancy analysis indicated that soils sampled from the different GMS treatments differentially influenced seedling biomass. A clone library of 267 soil bacteria was developed from sampled Orchard soils and Bioassay roots. These communities were dominated by Acidobacteria (25% of sequences), Actinobacteria (19%), δ-Proteobacteria (12%), β-Proteobacteria (10%), and these ratios differed among the GMS soils. Members of the family Comamonadaceae were detected only in tree-row soil, not in the Grass Lanes. The dominant sequences among 145 cloned fungi associated with apple seedling roots were Fusarium oxysporum (16% of sequences), an uncultured soil fungus submitted under DQ420986 (12%), and Rhodotorula mucilaginosa (9%). In a redundancy analysis, factors including fungal and oomycete community compositions, soil respiration rates, population sizes of culturable bacteria and fungi, soil organic matter content, and nutrient availability, were not significant predictors of apple seedling biomass in these soils. Different GMS treatments used by apple growers may influence subsequent ARD severity in replanted trees, but edaphic factors commonly associated with soil fertility may not reliably predict tree-root health and successful establishment of replanted orchards.     

The Effect of Glomus mosseae and Enterobacter aerogenes on Apple Seedlings Grown in Apple Replant Disease Soil

Inoculation of apple seedlings with the mycorrhizal fungus Glomus mosseae significantly increased their growth in apple replant disease soil that was neither pasteurized nor fertilized with monoammonium phosphate. The application of Enterobacter aerogenes and peat suppressed the stunting effect observed in apple replant disease soil. These results suggest the potential of using G. mosseae alone or a E.aerogenes and peat combination for the control of apple replant disease.     

Effects of nematodes,fungi and bacteria on the growth of young apple trees grown in apple replant disease soil

Growth, dry root weight of seedlings and root score of apple seedlings cv. McIntosh were reduced when soils were inoculated with Pratylenchus penetrans, Penicillium janthinellum, Constantinella terrestris, Trichoderma sp., and 4 strains of Bacillus subtilis. Trichoderma sp., and B-1 and B-26 strains of B. subtilis alone reduced plant growth but the combination of Trichoderma sp. + B. subtilis (B-1) and Trichoderma sp. + B. subtilis (B-26) increased plant height. Plant height, root weight and root score were significantly reduced when P. penetrans plus B. subtilis or P. penetrans plus fungi plus bacteria were present in the soil. It is suggested that fungi, bacteria, nematodes alone or their combinations such as nematodes plus bacteria or nematodes plus fungi plus bacteria may contribute towards the occurrence of apple replant disease.Contribution number 700.Contribution number 700.     

The effect of mode of application of Bacillus subtilis CA32r on control of Sclerotium rolfsii on Capsicum annuum

Abstract

Sclerotium rolfsii Sacc. the causal agent of collar and root rot of chili (Capsicum annuum L.), is one of the most important soil-borne pathogens in Sri Lanka. Bacillus subtilis CA32r, a stable spontaneous kanamycin resistant isolate, showing antagonism in a Petri plate assay, was selected for greenhouse pot experiments to control S. rolfsii. An ethyl acetate extract of the culture filtrate of B. subtilis CA32r inhibited radial colony growth as well as germination of sclerotia of the pathogen in vitro, indicating the presence of antifungal compound(s) in the culture extract. B. subtilis CA32r was investigated for its effectiveness as a biological control agent against S. rolfsii infecting transplanted chilli seedlings in greenhouse pot experiments. CA32r significantly decreased the disease incidence in terms of lesion development on stem base and roots depending on the mode of the bacterial application. Seed bacterization and soil application alone did not protect chilli plants, but root bacterization prior to the transplanting of seedlings significantly decreased the disease incidence caused by S. rolfsii. However, even in the combined treatment, seed bacterization and soil application, did not protect chilli plants. The best protection was achieved by combination of root bacterization prior to transplant and soil application of CA32r. Root bacterization resulted in maintaining higher numbers of bacteria at the collar region of chilli plants and may have shielded the most vulnerable area from the pathogen, resulting enhanced protection. Since the application of CA32r resulted in a significant reduction of the number of viable S. rolfsii propagules in the soil indicates that B. subtilis CA32r possesses not only protective but also eradicative potential.     

Effect of mycorrhizal inocula on the growth of Sitka spruce seedlings in different soils

Summary Different mycorrhizal fungi were tested for their effectiveness in promoting growth of Sitka spruce seedlings, in two contrasting soils, in a glasshouse pot experiment. In nursery soil,Laccaria amethystina significantly improved growth of seedlings in comparison toL. laccata. Seedlings inoculated with a forest isolate ofThelephora terrestris were significantly larger than those inoculated with a nursery isolate when grown in forest soil. The effectiveness ofComplexipes moniliformis in forest soil was poor in comparison to other mycorrhizal fungi. Strains aswell as species of mycorrhizal fungi affect seedling growth differently. These effects are further influenced by soil type.     

Evaluation of monoammonium phosphate and bacterial strains to increase tree growth and fruit yield in apple replant problem soil

The long-term effects of biological agents alone and in combination with monoammonium phosphate on tree growth and fruit production of apple trees planted on apple replant soil was studied for five years. Application of monoammonium phosphate (MAP) in the year of planting increased shoot growth, cross-sectional trunk area and fruit yield of McIntosh on M.26 rootstock for the first two years. The application of bacterial agents alone were not effective in increasing young tree growth except BACT-1 in 1987. None of the bacterial agents increased fruit yield when applied alone. The addition of certain bacterial agents to MAP application increased young tree growth in various years. The combination of bacterial agent B-10 and MAP reduced young tree growth and yield compared with the MAP treatment alone. These results suggest that the application of MAP alone may be sufficient to alleviate the replant problem and the addition of BACT-1, EBW-4 or B8 bacterial agents to this treatment may be beneficial to increase tree growth in some years. Contribution number 822. Contribution number 822.     

Characteristics and antimicrobial activity of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains isolated from soil

Antagonistic Bacillus strains were isolated from soil and analyzed for the purpose of determining whether they could be used as natural biological agents. Primary in vitro screening for antagonism of the isolates was performed against five phytopathogenic mould fungi. Strains TS 01 and ZR 02 exhibited the most pronounced inhibitory effects. They were identified as Bacillus subtilis on the basis of their morphological, cultural and physiology-biochemical properties as well as their hierarchical cluster analysis conducted by means of computer program SPSS. The antimicrobial activity of the strains from cultural medium and sterile filtrate were determined in vitro against a great number of predominantly phytopathogenic fungi and bacteria. TS 01 and ZR 02 strains exhibited very broad and at the same time degree varying antibiotic spectra of activities against both Gram-positive and Gram-negative microorganisms. Many of them were tested against sensitivity to the antimicrobial action of B. subtilis for the very first time. B. subtilis TS 01 and ZR 02 showed highest antifungal activity (sterile zone in diameter over 37 mm) against Alternaria solani, Botrytis cinerea, Monilia linhartiana 869, Phytophthora cryptogea 759/1 and Rhizoctonia sp. The most sensitive bacterial species were found to be Pseudomonas syringae pv. tomato Ro and Xanthomonas campestris with sterile zones 48.0 and 50.0 mm in diameter, respectively. The latter draws a conclusion that the isolated and identified Bacillus subtilis strains are promising natural biocontrol agents and should be further studied and tested for control of numerous plant diseases.     

Effects of Nitrogen and Phosphorus on the Growth of Microorganisms Associated with Apple Replant Disease and on Apple Seedlings Grown in Soil Infested with these Microorganisms*

The growth of fungi causing apple replant disease (ARD) was inhibited by the addition of N and P to the growing medium. The population of bacteria antagonistic to ARD-causing fungi was significantly increased in the growing medium supplemented with N 400 P 400 mg/l or greater. The application of nitrogen alone or in combination with phosphorus to soil infested with fungi or bacteria that cause apple replant disease significantly increased seedling height. The addition of P alone to these infested soils did not have any effect on seedling height. Significant increases in seedling height occurred with N applications when seedlings were grown in soil to which bacteria that are antagonistic to fungi causing ARD had been added. These results suggest that the application of N, with or without P, to apple replant soils may suppress the growth of ARD-causing fungi or bacteria and promote the growth of antagonistic bacteria.     

Experimental evidence of a deleterious soil microflora associated with Norway spruce decline in France and Germany

Norway spruce (Picea abies (L.) Karst.) seedlings were grown in a glasshouse pot experiment in soils from 11 declining and 7 healthy spruce stands from France and Germany. In soils from 9 declining stands, seedlings showed decline symptoms (needle yellowing). Soil pasteurization suppressed the symptoms, and reinoculation of the pasteurized soil with a rhizospheric extract from the corresponding stand re-induced yellowing. This suggests that a deleterious soil microflora is associated with spruce decline. The occurrence of this microflora seems to be correlated with the main chemical characteristics of the soils (low pH, low saturation of the adsorbing complex, low exchangeable Ca²⁺ and Mg²⁺, and high level of exchangeable Al). ei]R F Huettl     

Effects of isolates of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens that were dominant in soil from disturbed sites on growth of Betula platyphylla var. japonica seedlings

Effects of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens Melin (MRA) on growth of Betula platyphylla var. japonica seedlings were investigated under aseptic culture conditions. Three isolates of ectomycorrhizal fungi and two isolates of MRA were used. One MRA isolate was Phialocephala fortinii. Previous field work revealed that these isolates were dominant on the roots of B. platyphylla var. japonica seedlings grown in a mineral subsoil that had been exposed by the removal of surface soil. After a 100-day incubation, the growth of the seedlings was significantly enhanced by the colonization of these ectomycorrhizal fungal isolates as compared with uninoculated seedlings. In contrast, the growth of seedlings was retarded by the colonization of the MRA isolates. The growth of seedlings that were co-inoculated with ectomycorrhizal fungi and MRA was similar to that of uninoculated seedlings in most cases. These results suggest that ectomycorrhizal fungi have a beneficial effect on the growth of B. platyphylla var. japonica seedlings and that they suppress the deleterious effect of MRA. Thus, these ectomycorrhizal fungi probably have an important role in establishing B. platyphylla var. japonica seedlings during the initial stage of re-vegetation following site disturbance by the removal of surface soil.     

Resident bacteria, nitric oxide emission and particle size modulate the effect of Brassica napus seed meal on disease incited by Rhizoctonia solani and Pythium spp

Amendment of orchard soil with low-glucosinolate Brassica napus (rape) seed meal (RSM) suppresses infection of apple roots by Rhizoctonia solani but increases incidence of Pythium spp. infection. Following incorporation of Brassica sp. seed meals, soils were monitored for changes in populations of selected saprophytic and plant pathogenic microorganisms. When conducted in pasteurized soil, which possessed high numbers of Bacillus spp. and lower than detectable numbers of Streptomyces spp., RSM amendment did not provide control of R. solani. Populations of streptomycetes in RSM-amended soil increased to stable levels >20-fold higher than in non-amended soil. Disease suppressiveness was restored to pasteurized RSM-amended soil by adding any of several Streptomyces strains. Maximal rates of nitrification in orchard soil, determined by nitric oxide emission, were observed within two weeks following RSM amendment and inhibition of nitrification via application of nitrapyrin abolished the capacity of RSM to suppress R. solani infection of apple roots when seedlings were planted one day after soil amendment. Apple seedling mortality and Pythium spp. root infection were highest for seedlings planted immediately following incorporation of B. napus cv. Athena RSM, particularly when meal was added in a flake rather than powder form. Lower infection frequencies were observed for seedlings planted four weeks after RSM incorporation, even for soil in which densities of culturable Pythium spp. had not declined. Our results demonstrate that suppression of Rhizoctonia root rot in response to RSM amendment requires the activity of the resident soil microbiota and that initial disease control is associated with the generation of nitric oxide through the process of nitrification.     

The effect of soil pH on the ability of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill.

We examined the effect of two levels of soil pH (5 and 6) on the ability (effectiveness) of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill. at a deficient supply of P. Plants were inoculated with one of six fungal isolates [Laccaria laccata (Scop. ex Fr.) Berk. and Br. (isolates A and B), Pisolithus tinctorius (Pers.) Coker and Couch (isolates A and B), Descolea maculata Bough. and Mal. and Setchelliogaster sp. nov.] and were grown in a P-deficient sand, in pots, in a temperature-controlled glasshouse. Seedlings were harvested 89 days after planting and were assessed for dry matter production, tissue P concentrations, ectomycorrhizal colonization of roots and hyphal development in soil.Uninoculated plants had less than 5% of their fine root length colonized by ectomycorrhizal fungi. In contrast, inoculated plants had 30% or greater of their fine root length ectomycorrhizal. Inoculation increased the uptake of P and growth of plants for all isolates and at both levels of soil pH, although growth responses to inoculation were greater at pH 6, particularly for the two L. laccata isolates. Isolates which colonized roots most extensively increased plant growth to the greatest extent. D. maculata was the most effective fungal isolate at pH 5, and both D. maculata and L. laccata A were most effective at pH 6. The effects of soil pH on plant growth were also related to some extent to the effects of soil pH on colonized root length. Growth responses to inoculation were related less well to hyphal development in soil. The L. laccata isolates formed more hyphae in soil (on a per pot, per m of fine root, and per m of colonized fine root basis) than other fungal isolates, but were not always more effective in increasing plant grown.     

Biological control of postharvest fungal rot of yam (<Emphasis Type="Italic">Dioscorea</Emphasis> spp.) with<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The potential of isolates of Bacillus subtilis from yam farm soil to control rot of yam in storage barns was investigated. Yam tubers inoculated in vivo with B. subtilis showed no rot while those inoculated with Aspergillus niger, Botryodiploidia theobromae or Penicillium oxalicum showed considerable rot. The set of yams in which B. subtilis and the fungi were simultaneously inoculated produced rot whereas those in which B. subtilis was inoculated a day before the fungi was inoculated were totally reduced or free of rot. Many fewer fungi were isolated from the surface of tubers treated with B. subtilis than from the untreated (control) and there was high recovery of B. subtilis (99–100%) throughout the period of storage. Rot build up was faster in uninoculated control tubers or those inoculated with a spoilage fungus, while those treated with the antagonist were totally reduced or free of rot. The culture filtrate of B. subtilis prevented spore germination in some spoilage fungi. The importance of this study in relation to farmers in developing countries is discussed.     

Biological control of pre-harvest aflatoxin contamination in groundnut (Arachis hypogaea L.) with Bacillus subtilis G1

The antagonistic activity of Bacillus subtilis strain G1 was tested against various isolates of Aspergillus flavus in vitro. A talc-based powder formulation of B. subtilis strain G1 was prepared and evaluated to control A. flavus infection and aflatoxin B1 contamination in groundnut under greenhouse and field conditions. The results showed that B. subtilis strain G1 could inhibit the growth of all isolates of A. flavus tested in dual culture assay and the growth inhibition ranged from 93 to 100%. Results of greenhouse and field experiments indicated that B. subtilis strain G1 when applied to groundnut as seed treatment and soil application significantly suppressed A. flavus population in the soil, A. flavus infection and aflatoxin B1 content in kernels and increased the pod yield. These studies show that B. subtilis strain G1 has potential as a biocontrol agent for control of aflatoxin contamination in groundnut.     

Tunisian isolates of Trichoderma spp. and Bacillus subtilis can control Botrytis fabae on faba bean

Faba bean crops worldwide are often attacked by different diseases, particularly chocolate spot caused by Botrytis fabae Sard. Fungal and bacterial isolates collected from faba bean and barley leaves in Tunisia were evaluated for their antagonistic potential against B. fabae. In a test on detached leaves, the highest rate of decrease in disease severity was scored by Trichoderma viride, followed by T. harzianum, the fungicide Carbendazim then Bacillus subtilis. Under glasshouse conditions, all tested fungi resulted in significant disease severity reduction. T. viride reduced the rate of chocolate spot infestation on leaves and stems by 35% and 31.5%, respectively, when the rate on the control was 100%. For T. harzianum, Carbendazim and B. Subtilis, the rates of infestation on the leaves were 41.7%, 43.1% and 59.7%, respectively. On the stems, T. harzianum scored the lowest rate of 54.2% followed by B. subtilis with 79.2% then Carbendazim with 87.5%. Two consecutive seasons of field trials using the Trichoderma species, B. subtilis and Carbendazim showed significant and consistent reduction in the severity of chocolate spot infestation rates. The highest protection against the disease was obtained from T. viride. Based on these results, Tunisian isolates of Trichoderma spp. can be recommended for developing commercial bio-fungicides for integrated management of chocolate spot.     

Mycorrhizal Fungi Prevent Disease in Stressed Pine Seedlings

The common rhizospheric fungus Cylindrocarpon destructans caused severe root damage leading to mortality in light-starved, stunted seedlings of Pinus sylvestris. This effect was, however, almost fully prevented by the presence of mycorrhizal fungi; i.e. Laccaria laccata, Pisolithus tinctorius, or unidentified species present in small amounts of conifer soil. Protection took place even before visible mycorrhizae were found on the roots. This is the first time that protection ofstressed plants against non-parasitic (non-invasive) pathogens has been reported. The results indicate that root protection by mycorrhizal fungi may not only be effective inside the root but probably also extends out into the rhizoplane or rhizosphere.     

Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis

In this work, the potential of Bacillus subtilis strain M4 at protecting plants against fungal diseases was demonstrated in different pathosystems. We provide evidence for the role of secreted lipopeptides, and more particularly of fengycins, in the protective effect afforded by the strain against damping-off of bean seedlings caused by Pythium ultimum and against gray mold of apple in post-harvest disease. This role was demonstrated by the strong biocontrol activity of lipopeptide-enriched extracts and through the detection of inhibitory quantities of fengycins in infected tissues. Beside such a direct antagonism of the pathogen, we show that root pre-inoculation with M4 enabled the host plant to react more efficiently to subsequent pathogen infection on leaves. Fengycins could also be involved in this systemic resistance-eliciting effect of strain M4, as these molecules may induce the synthesis of plant phenolics involved in or derived from the defense-related phenylpropanoid metabolism. Much remains to be discovered about the mechanisms by which Bacillus spp suppress disease. Through this study on strain M4, we reinforce the interest in B. subtilis as a pathogen antagonist and plant defense-inducing agent. The secretion of cyclic fengycin-type lipopeptides may be tightly related to the expression of these two biocontrol traits.     

Effects of Pythium species on the growth of apple and their possible causal role in apple replant disease

Several lines of circumstantial evidence collectively indicated that poor early growth of apple (‘replant disease’) might be associated with the effects of soil-borne pythiaceous fungi. This hypothesis was supported by pathogenicity tests. All isolates tested of P. sylvaticum and certain isolates of seven other Pythium spp. significantly reduced the growth of apple seedlings. The growth reductions caused by certain Pythium isolates were of comparable magnitude to the growth increases occurring after chloropicrin-fumigation of apple orchard soils. The Pythium isolates most virulent to apple were of low virulence to a clonal cherry rootstock. Reappraisal of the nature of the disease as a non-specific soil malaise is consistent with established features of the pathology of Pythium spp. The disease, however, is an ill-defined ‘poor growth phenomenon’ with no diagnostic symptoms and conclusive evidence that Pythium spp. are widely causal is likely to be elusive.     

Impact of Bacillus subtilis JA, a biocontrol strain of fungal plant pathogens, on arbuscular mycorrhiza formation in Zea mays

Summary  In the present study, the influence of Bacillus subtilis JA on arbuscular mycorrhizal fungi (AMF) was evaluated by either pot culture or in vitro conditions, respectively. Under the pot culture conditions, the inoculation of B. subtilis JA decreased the frequency (% F) of the root colonization by indigenous arbuscular mycorrhizal fungi and the shoot dry weight of maize (Zea mays L.), but had no apparent effect on the intensity (% I) of AM fungal root colonization. The unknown volatile emitted from the B. subtilis JA in vitro significantly inhibited spore germination and the hyphal growth in the dual-compartment experiments. Moreover, the data from the direct interaction between B. subtilis JA and Glomus etunicatum showed that soluble antifungal lipopeptides influenced the development of AMF. Therefore, the application of antifungal Bacillus strains should take the compatibility with the indigenous beneficial fungi into consideration.