Cloning and expression of an antifungal chitinase gene of a novel Bacillus subtilis isolate from Taiwan potato field

A chitinase producing Bacillus subtilis CHU26 was isolated from Taiwan potato field. This strain exhibited a strong extra-cellular chitinase activity on the colloidal chitin containing agar plate, and showed a potential inhibit activity against phytopathogen, Rhizoctonia solani. The gene encoding chitinase (chi18) was cloned from the constructed B. subtilis CHU26 genomic DNA library. The chi18 consisted of an open reading frame of 1791 nucleotides and encodes 595 amino acids with a deduced molecular weight of 64kDa, next to a promoter region containing a 9 base pair direct repeat sequence (ATTGATGAA). The deduced amino acid sequence of the chitinase from Bacillus subtilis CHU26 exhibits 62% and 81% similarity to those from B. circulans WL-12 and B. licheniformis, respectively. Subcloned chi18 into vector pGEM3Z and pYEP352 to construct recombinant plasmid pGCHI18 and pYCHI18, respectively, chitinase activity could be observed on the colloidal chitin agar plate from recombinant plasmid containing Escherichia coli transformant. Cell-free culture broth of pYCHI18 containing E. coli transformant decreased R. solani pathogenic activity more than 90% in the antagonistic test on the radish seedlings (Raphanus sativus Linn.).     

Biocontrol of Rhizoctonia solani and Pythium ultimum on Capsicum by Trichoderma koningii in potting medium.

Two isolates of Trichoderma koningii were evaluated for efficacy in control of damping-off diseases in seedlings of Capsicum annuum grown in pasteurized potting medium in a glasshouse. A selected isolate of binucleate Rhizoctonia and two fungicides were also included as standards for control of Rhizoctonia solani and Pythium ultimum var. sporangiiferum. Both isolates of T. koningii reduced seedling death caused by R. solani in one of two experiments, and by P. u. sporangii-ferum in two of three experiments. Neither isolate of T. koningii suppressed damping-off caused by either pathogen as consistently as the binucleate Rhizoctonia or fungicides. The implications of these results for commercial disease management are discussed.     

Biocontrol of Rhizoctonia solani Damping-Off of Tomato with Bacillus subtilis RB14

Bacillus subtilis RB14, which showed antibiotic activities against several phytopathogens in vitro by producing the antibiotics iturin A and surfactin, was subjected to a pot test to investigate its ability to suppress damping-off of tomato seedlings caused by Rhizoctonia solani. To facilitate recovery from soil, B. subtilis RB14-C, a spontaneous streptomycin-resistant mutant of RB14, was used. Damping-off was suppressed when the culture broth, cell suspension, or cell-free culture broth of RB14-C was inoculated into soil. Iturin A and surfactin were recovered from the soils inoculated with the cell suspension of RB14-C, confirming that RB14-C produced them in soil. The gene lpa-14, which was cloned from RB14 and required for the production of both antibiotics, was mutated in RB14-C, and a mutant, R(Delta)1, was constructed. The level of disease suppressibility of R(Delta)1 was low, but R(Delta)1(pC115), a transformant of R(Delta)1 with the plasmid pC115 carrying lpa-14, was restored in suppressibility. These results show that the antibiotics iturin A and surfactin produced by RB14 play a major role in the suppression of damping-off caused by R. solani. RB14-C, R(Delta)1, and R(Delta)1(pC115) persisted in soil during the experimental period and were recovered from the soil, mostly as spores.     

Amendment with peony root bark improves the biocontrol efficacy of Trichoderma harzianum against Rhizoctonia solani

We tested Trichoderma harzianum as a biocontrol agent for Rhizoctonia solani AG2-1, using six natural antifungal materials to improve its efficacy. Among the six materials tested, peony (Paeonia suffruticosa) root bark (PRB) showed the strongest antifungal activity against R. solani AG2-1, and was not antagonistic to T. harzianum. Scanning electron microscopy showed that treatment with PRB extract resulted in shortened and deformed R. solani AG2-1 hyphal cells. The control of radish damping-off caused by R. solani AG2-1 was greatly increased by combined treatments of T. harzianum and PRB, as compared with either of the two treatments alone, with the control effect increased from 42.3-51.5% to 71.4-87.6%. The antifungal compound in PRB, which was isolated in chloroform and identified as paeonol by mass spectrometry, 1H NMR, and 13C NMR analyses, inhibited the growth of R. solani AG2-1 but not that of T. harzianum. Thus, PRB powder or extract may be used as a safe additive to T. harzianum to improve the control of the soil borne diseases caused by R. solani AG2-1.     

Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.     

Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.     

Soil suppressiveness to Rhizoctonia solani and microbial diversity

Rhizoctonia solani anastomosis group 2-2IIIB causes damping-off, black root rot and crown rot in sugar beet (Beta vulgaris). Based on experiences of growers and field experiments, soils can become suppressive to R. solani. The fungus may be present in the soil, but the plant does not show symptoms. Understanding the mechanisms causing soil suppressiveness to R. solani is essential for the development of environmentally friendly control strategies of rhizoctonia root rot in sugar beet. A bioassay that discriminates soils in their level of disease suppressiveness was developed. Results of bioassays were in accordance with field observations. Preliminary results indicate an active role of microbial communities. Our research is focused on the disentanglement of biological mechanisms causing soil suppressiveness to R. solani in sugar beet. Therefore, we are handling a multidisciplinary approach through experimental fields, bioassays, several in vitro techniques and molecular techniques (PCR-DGGE).     

Detoxification of oxalic acid by pseudomonas fluorescens strain pfMDU2: implications for the biological control of rice sheath blight caused by Rhizoctonia solani

Rhizoctonia solani isolates varying in their virulence were tested for their ability to produce oxalic acid (OA) in vitro. The results indicated that the virulent isolates produced more OA than the less virulent isolates. In order to isolate OA-detoxifying strains of Pseudomonas fluorescens, rhizosphere soil of rice was drenched with 100 mM OA and fluorescent pseudomonads were isolated from the OA-amended soil by using King's medium B. These isolates were tested for their antagonistic effect towards growth of R. solani in vitro. Among them P. fluorescens PfMDU2 was the most effective in inhibiting the mycelial growth of R. solani. P. fluorescens PfMDU2 was capable of detoxifying OA and several proteins were detected in the culture filtrate of PfMDU2 when it was grown in medium containing OA. To investigate whether the gene(s) involved in OA-detoxification resides on the plasmids in P. fluorescens PfMDU2, a plasmid-deficient strain of P. fluorescens was generated by plasmid curing. The plasmid-deficient strain (PfMDU2P-) failed to grow in medium containing OA and did not inhibit the growth of R. solani. Both PfMDU2 and PfMDU2P- were tested for their efficacy in controlling sheath blight of rice under greenhouse conditions. Seed treatment followed by soil application of rice with P. fluorescens strain, PfMDU2, reduced the severity of sheath blight by 75% compared with the control, whereas PfMDU2P- failed to control sheath blight disease.     

Role of chitinase and beta-1,3-glucanase activities produced by a fluorescent pseudomonad and in vitro inhibition of Phytophthora capsici and Rhizoctonia solani

A study was conducted to investigate the possibility of involvement of chitinase and beta-1,3-glucanase of an antagonistic fluorescent Pseudomonas in growth suppression of phytopathogenic fungi, Phytophthora capsici and Rhizoctonia solani. Fluorescent Pseudomonas isolates GRC(3) and GRC(4) were screened for their antifungal potential against phytopathogenic fungi by using dual culture technique both on solid and liquid media. The percent inhibition was calculated. Various parameters were monitored for optimization of enzyme activities by fluorescent Pseudomonas GRC(3). The involvement of chitinases, beta-1,3-glucanases, and antifungal metabolites of nonenzymatic nature was correlated with the inhibition of P. capsici and R. solani. The results provide evidence for antibiosis as a mechanism for antagonism. The study also confirms that multiple mechanisms are involved in suppressing phytopathogens as evidenced by the involvement of chitinase and beta-1,3-glucanase in inhibition of R. solani but not P. capsici by isolate GRC3.     

Fungitoxicity of mineral oils against Rhizoctonia solani causing damping-off of mung bean (Phaseolus aureus)

Three mineral oils, BSO, EWOS and E9267 and one vegetable oil (mustard oil), did not appreciably inhibit the mycelial growth of Rhizoctonia solani. However, treatment of 100 g seeds of mung bean with 2 ml EWOS and E9267 oils controlled more than 90% of the pre- and post-emergence damping-off and protected seedlings in soil inoculated with R. solani 5 days after sowing. Soaking seeds in solutions of these oils or drenching the soil did not control damping-off. Mustard oil controlled only pre-emergence damping-off.     

Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots

AIMS: To isolate endophytic Streptomyces strains from tomato and examine their antimicrobial activity. METHODS: Endophytic Streptomyces strains were isolated using surface-sterilization methods and identified by morphological characteristics. Antimicrobial activities were measured by the agar plate sensitivity method. Antifungal activity in vivo was measured by seedling mortality in infested soils. RESULTS: Twenty-one per cent of endophytic streptomycete isolates produced antibacterial metabolites and 41% produced antifungal metabolites in S medium. Sixty-five per cent of the most frequently isolated strains inhibited the growth of Rhizoctonia solani by the antibiosis assay but only 32% produced metabolites active against R. solani in S medium. Growth promotion and enhanced disease resistance of seedlings inoculated with Streptomyces sp. strain S30 were observed in tomato but not in cucumber seedlings. CONCLUSIONS: Endophytic Streptomyces spp. strains were successfully isolated using stringent methods and strain S30 promoted growth and enhanced resistance to R. solani in tomato seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic streptomycetes showing antifungal activity in vitro and in vivo may indicate the potential for their use as biocontrol agents particularly of R. solani disease of tomato seedlings.     

Processed Manure as Carrier to Introduce Trichoderma harzianum: Population Dynamics and Biocontrol Effect on Rhizoctonia solani

Manure pellets produced from processed swine faeces can be used as carrier material for the biocontrol fungus Trichoderma harzianum. The antagonist can grow and sporulate on the processed manure powder as the sole source of carbon and nutrients. The incorporation of conidia in pellets of the processed manure was shown to be feasible on a laboratory scale. Survival of the fungus in the pellets during storage was satisfactory. The population dynamics of T. harzianum were studied using a benomyl-resistance marker after introduction of conidia into soil. The antagonist could colonize and spread through a number of non-sterile soils and was able to establish a stable population over a period exceeding 125 days. Under sterile conditions, the propagation of T. harzianum in soil was much greater than under non-sterile conditions. The incorporation of antagonist conidia in pellets was found to be essential for the successful colonization of non-sterile soil. In growth chamber experiments, application of T. harzianum via processed manure pellets reduced damping-off of sugar beet seedlings caused by Rhizoctonia solani in artificially and naturally infested soil. In artificially infested soil, T. harzianum reduced the population of R. solani and protected beet seedlings from damping-off 3 weeks after introduction. The application of T. harzianum to naturally infested soil increased the number of healthy beet seedlings more than two-fold.     

Solid formulations of binucleate Rhizoctonia isolates suppress Rhizoctonia solani and Pythium ultimum in potting medium

Two isolates of binucleate Rhizoctonia spp., previously selected for efficacy in suppression of Rhizoctonia solani and Pythium spp., as well as plant growth promotion, were incorporated into various solid substrate formulations. These formulated products were assayed at three doses in three glass-house experiments for biocontrol of damping-off diseases in Capsicum annuum. R. solani anastomosis group 4 or Pythium ultimum var. sporangiiferum were incorporated into pasteurized potting medium with each formulated binucleate Rhizoctonia product. All formulations were effective against both pathogens in at least two experiments, but some formulations of one isolate of binucleate Rhizoctonia did not give consistent control of R. solani in one experiment. The most consistent formulation, which provided control of both pathogens at all doses of binucleate Rhizoctonia, was the simple substrate of rice hulls. The implications for commercialization of a biocontrol product are discussed.     

Pseudomonas fluorescens DR54 Reduces Sclerotia Formation,Biomass Development,and Disease Incidence of Rhizoctonia solani Causing Damping-Off in Sugar Beet

Effects of the biocontrol strain, Pseudomonas fluorescens DR54, on growth and disease development by Rhizoctonia solani causing damping-off in sugar beet were studied in soil microcosms and in pot experiments with natural, clay-type soil. In pot experiments with P. fluorescens DR54-treated seeds, significantly fewer Rhizoctonia-challenged seedlings showed damping-off symptoms than when not inoculated with the biocontrol agent. In the rhizosphere of P. fluorescens DR54 inoculated seeds, the bacterial inoculant was present in high numbers as shown by dilution plating and immunoblotting. By the ELISA antibody technique and direct microscopy of the fungal pathogen grown in soil microcosms, it was shown that the presence of P. fluorescens DR54 on the inoculated seeds had a strong inhibitory effect on development of both mycelium biomass and sclerotia formation by R. solani. In the field experiment, plant emergence was increased by treatment with P. fluorescens DR54 and the inoculant was found to be the dominating rhizosphere colonizing pseudomonad immediately after seedling emergence.     

Effect of Herbicides on the Toxicity of Fungicides Against Rhizoctonia solani Causing Damping-off of Cotton

The effect of two herbicides (paraquat and simazine on the antifungal activity of two fungicides (captan and mounsrin) against Rhizoctonia solani was studied. when the herbicides paraquat and simazine were applied to soil they altered the effectiveness of both fungicides in controlling R. solani , thus causing damping-off of cotton. Both herbicides increased the toxicity of both fungicides against mycelial growth of the fungus. In pot tests, seeds or soil treated with captan or mounsrin, gave better control of R. solani damping-off disease when the soil was treated with paraquat or simazine compared to untreated soil. Captan was, however, found to be more effective in controlling the disease than mounsrin.     

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.     

Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by endophytic actinomycetes isolated from native plants of Algerian Sahara

Thirty-four endophytic actinomycetes were isolated from the roots of native plants of the Algerian Sahara. Morphological and chemical studies showed that twenty-nine isolates belonged to the Streptomyces genus and five were non-Streptomyces. All isolates were screened for their in vitro antifungal activity against Rhizoctonia solani. The six that had the greatest pathogen inhibitory capacities were subsequently tested for their in vivo biocontrol potential on R. solani damping-off in sterilized and non-sterilized soils, and for their plant-growth promoting activities on tomato seedlings. In both soils, coating tomato seeds with antagonistic isolates significantly reduced (P < 0.05) the severity of damping-off of tomato seedlings. Among the isolates tested, the strains CA-2 and AA-2 exhibited the same disease incidence reduction as thioperoxydicarbonic diamide, tetramethylthiram (TMTD) and no significant differences (P < 0.05) were observed. Furthermore, they resulted in a significant increase in the seedling fresh weight, the seedling length and the root length of the seed-treated seedlings compared to the control. The taxonomic position based on 16S rDNA sequence analysis and phylogenetic studies indicated that the strains CA-2 and AA-2 were related to Streptomyces mutabilis NBRC 12800^T (100% of similarity) and Streptomyces cyaneofuscatus JCM 4364^T (100% of similarity), respectively.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Behaviour of the fungus Rhizoctonia solani Kiihn in the soil

Rhizoctonia solani was found able to grow as a saprophyte through natural unsterilized soil. Its rate of growth under different soil conditions in glass tumblers was studied by the Rossi-Cholodny soil-plate method. Growth was most rapid at the lowest soil-moisture content tested, viz. 30 % saturation, and was accelerated by forced aeration of the soil. The maximum distance to which mycelial growth could be supported on the food reserves of the agar inoculum alone was some 5 cm., as shown by extent of growth through tubes of moist sand, but in 23 days the fungus grew 21–24 cm through tubes of soil. Removal of the agar disk 2 days after inoculation of the tubes reduced growth through sand by more than half, but through soil by only a small proportion. In soil, Rhizoctonia was able to cause 100% damping-off of radish seedlings planted at a radial distance of 4 cm. from the agar inoculum, and some 40 % damping-off at a distance of 9 cm. The depressing effect of additions of 1 % ground-wheat straw or dried grass to the soil upon growth of the fungus was attributed to (1) the negligible cellulose-decomposing ability of Rhizoctonia, (2) nitrogen starvation of the mycelium, through rapid utilization of the available soil nitrogen by the cellulose-decomposing micro-organisms multiplying upon the fresh organic material, (3) fungistatic action on Rhizoctonia of the respiratory carbon dioxide produced by the cellulose-decomposers.     

Early induction of the Arabidopsis GSTF8 promoter by specific strains of the fungal pathogen Rhizoctonia solani

The Arabidopsis glutathione S-transferase GSTF8 promoter directs root-specific responses to stress. In this study, the response of this promoter to plant infection with Rhizoctonia solani was investigated using a luciferase reporter system. Arabidopsis seedlings harboring the GSTF8:luciferase construct were monitored in vivo for bioluminescence following infection with R. solani. Although the reporter gene was induced in infected roots, the response differed markedly between R. solani strains and was not observed with aggressive strains that caused death of the seedlings. The three strains tested in detail progressed through typical stages of infection, but ZG1-1 induced the GSTF8 promoter in most seedlings, ZG3 induced it in approximately 25% of seedlings, and ZG5 caused little response. Induction of specific root segments occurred early in the infection process in root regions with very limited mycelium visible. In root segments with substantial mycelium, GSTF8 promoter activity no longer was observed. Induction by ZG1-1 also was observed in plants harboring a tetramer of the ocs element from the GSTF8 promoter, suggesting that this element helps mediate the response. Crossing GSTF8:luciferase plants with plants harboring an Nah-G construct that degrades salicylic acid did not abolish the response, indicating that the GSTF8 promoter response to R. solani may be mediated by signals other than salicylic acid.