一株抗茶炭疽菌和魔芋镰刀菌的淀粉酶产色链霉菌的分离、鉴定及生防潜能

【背景】放线菌是一类重要的生防菌,具有强大的代谢活性,能产生抗生素、酶、酶抑制剂和激素等天然产物抑制病原物生长。【目的】从茶树根际分离得到放线菌,研究候选放线菌对茶炭疽菌和魔芋镰刀菌的抑菌活性及其生防潜能。【方法】分别以茶炭疽病致病菌Colletotrichum camelliae和魔芋茎腐病致病菌Fusarium solani为指示菌,采用土壤稀释涂布法、平板对峙法和菌丝生长速率法,从茶树根际土壤中分离、筛选拮抗放线菌,并根据菌株的形态特征、生理生化特性和系统发育分析结果对其进行分类鉴定,并开展候选放线菌的产促生相关物质和分泌细胞壁水解酶能力的定性检测试验。【结果】共分离得到14株拮抗放线菌,菌株A-dyzsc04-2的拮抗效果最强,被鉴定为淀粉酶产色链霉菌（Streptomyces diastatochromogenes）。该菌株的活菌体对C. camelliae和F. solani的抑制率分别为66.71%±1.23%和71.59%±2.46%,其无菌发酵滤液对2种指示菌的抑菌率均大于90%;此外,菌株A-dyzsc04-2还具有产嗜铁素和葡聚糖酶以及溶解无机磷的能力。【结论】菌株A-dyzsc04-2是一株优良的生防菌,具有较高的开发利用价值,研究结果为菌株A-dyzsc04-2防治茶炭疽病和魔芋茎腐病提供了理论支撑。     

Proteomic response of Rhizoctonia solani GD118 suppressed by Paenibacillus kribbensis PS04

Rice sheath blight, caused by Rhizoctonia solani, is considered a worldwide destructive rice disease and leads to considerable yield losses. A bio-control agent, Paenibacillus kribbensis PS04, was screened to resist against the pathogen. The inhibitory effects were investigated (>80 %) by the growth of the hyphae. Microscopic observation of the hypha structure manifested that the morphology of the pathogenic mycelium was strongly affected by P. kribbensis PS04. To explore essentially inhibitory mechanisms, proteomic approach was adopted to identify differentially expressed proteins from R. solani GD118 in response to P. kribbensis PS04 using two-dimensional gel electrophoresis. Protein profiling was used to identify 13 differential proteins: 10 proteins were found to be down-regulated while 3 proteins were up-regulated. These proteins were involved in material and energy metabolism, antioxidant activity, protein folding and degradation, and cytoskeleton regulation. Among them, material and energy metabolism was differentially regulated by P. kribbensis PS04. Protein expression was separately inhibited by the bio-control agent in oxidation resistance, protein folding and degradation, and cytoskeleton regulation. Proteome changes of the mycelium assist in understanding how the pathogen was directly suppressed by P. kribbensis PS04.     

Isolation and evaluation of the biocontrol potential of Talaromyces spp. against rice sheath blight guided by soil microbiome

Rice sheath blight caused by Rhizoctonia solani is the major disease of rice that seriously threatens food security worldwide. Efficient and eco-friendly biological approaches are urgently needed since no resistant cultivars are available. In this study, fallow and paddy soils were initially subjected to microbiome analyses, and the results showed that Talaromyces spp. were significantly more abundant in the paddy soil, while Trichoderma spp. were more abundant in the fallow soil, suggesting that Talaromyces spp. could live and survive better in the paddy soil. Five Talaromyces isolates, namely, TF-04, TF-03, TF-02, TF-01 and TA-02, were isolated from the paddy soil using sclerotia of R. solani as baits and were further evaluated for their activity against rice sheath blight. These isolates efficiently parasitized the hyphae and rotted the sclerotia even at higher water contents in the sterilized sand and the soil. Isolate TF-04 significantly promoted rice growth, reduced the severity of rice sheath blight and increased the rice yield under outdoor conditions. Defence-related genes were upregulated and enzyme activities were enhanced in rice treated with isolate TF-04. Our research supplies a microbiome-guided approach to screen biological control agents and provides Talaromyces isolates to biologically control rice sheath blight.     

利用纤维二糖差向异构酶制备乳果糖的研究进展

乳果糖是由D-半乳糖和D-果糖两个基团通过β-1,4糖苷键连接而成的还原型二糖;乳果糖口服液具有治疗慢性便秘和肝性脑病的功效,在100多个国家作为常见非处方药(OTC)使用,需求量十分巨大;乳果糖还可以作为益生元改善人体肠道菌群关系。乳果糖的生产依赖化学法,其催化剂对人体有害,下游分离难度大。近年来,纤维二糖差向异构酶被发现能够高效催化乳糖制备乳果糖,该技术绿色环保、步骤简单,具有很强的产业化前景。本文结合自身研究经历对纤维二糖差向异构酶的研发情况进行总结,并综述了乳果糖酶法制备技术的现状。     

Anti-fungal activity of essential oils of Ceylon Eucalyptus species for the control of Fusarium solani and Sclerotium rolfsii

The essential oils were extracted from the leaves of Eucalyptus microcorys, Eucalyptus grandis and Eucalyptus robusta which were grown in Sri Lanka and their major chemical compounds were determined. 1,8-Cineole and α-pinene were identified as major aroma compounds in these oils. In this study, the anti-fungal activity of essential oils of E. microcorys, E. grandis and E. robusta, ethanol extract of E. microcorys and 1,8-cineole were evaluated against Sclerotium rolfsii, a fungi responsible for leaf spot disease of indoor plants and Fusarium solani, a fungi responsible for dry rot diseases of potato by poisoned food technique, and minimum inhibitory concentrations (MICs) were determined. The essential oils from three Eucalyptus species showed significant inhibitory effect against S. rolfsii and F. solani than the ethanol extract of E. microcorys. Of treatments, the essential oil of E. grandis showed the best anti-fungal activity with the MIC values of less than 0.1% for S. rolfsii and 0.5% for F. solani. The MICs of the oils of E. microcorys and E. robusta were between 0.3–0.5% against S. rolfsii and 0.5–0.75% for F. solani. The 1,8-cineole did not exhibit inhibition activity as much of Eucalyptus essential oils and hence, it can be assumed that minor chemical components of the oils contribute to the growth inhibition of the tested fungi. This is the first report of anti-fungal activity of Sri Lankan oils of E. microcorys, E. grandis and E. robusta and ethanol extract of E. microcorys against S. rolfsii and F. solani. These findings would be useful for the designing of natural fungicide for agriculture- and food-based industries.     

林下参内生球毛壳菌FS-01对人参病原真菌的抑制作用

为探究林下参内生真菌球毛壳菌(Chaetomium globosum)FS-01菌株对人参病原菌的抑菌作用,该研究在实验室条件下,测定了FS-01菌株菌丝、发酵液和孢子悬浮液对人参黑斑病菌(Alternaria panax)、人参菌核病菌(Sclerotinia schinseng)、人参灰霉病菌(Botrytis cinerea)、人参立枯病菌(Rhizoctonia solani)、人参根腐病菌(Fusarium solani)5种人参病原菌的抑制作用。结果表明:内生真菌球毛壳菌FS-01对5种病原菌均有抑制作用,其中,对人参黑斑病菌的抑制作用最高,为30.80%,其次是人参立枯病菌、人参菌核病菌、人参根腐病菌和人参灰霉病菌; 发酵液抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株发酵液的PDA培养基上,对人参灰霉病菌的抑制作用最高,为82.09%,其次是人参菌核病菌、人参黑斑病菌、人参立枯病菌和人参根腐病菌; 孢子抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株孢子悬浮液的PDA培养基上,对人参黑斑病菌的抑制作用最高,为83.72%,其次是人参灰霉病菌、人参立枯病菌、人参菌核病菌和人参根腐病菌。综上结果认为,内生真菌球毛壳菌FS-01菌株对人参病原菌均有很高的抑菌作用,可作为人参病原菌的生防菌株资源。     

Zerumbone: a potential fungitoxic agent isolated from Zingiber cassumunar Roxb.

The rhizomes of Zingiber cassumunar exhibited strong fungitoxic action against Rhizoctonia solani, the damping-off pathogen. On chemical and spectral investigations, the antifungal compound was found to be zerumbone — a sesquiterpene. Its minimum effective dose against R. solani was 1000 ppm, much lower than some commercial fungicides. Zerumbone had fungistatic activity, a narrow fungitoxic spectrum and was not phytotoxic. Moreover, when used as a seed treatment, zerumbone could control damping-off disease of Phaseolus aureus caused by Rhizoctonia solani by 85.7%.     

Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays

Two leaf disc bioassays were developed for screening bacteria as putative biological control agents of Botrytis cinerea and Rhizoctonia solani on lettuce. Aerobic spore and non‐spore forming bacteria were isolated from the phylloplane, rhizoplane and rhizosphere of symptom‐free lettuce plants grown in the presence and absence of chitin or composted bark soil amendments. Bacteria, previously isolated from other plants, were also included in the primary screen initially against B. cinerea. One hundred and twenty‐seven of 700 isolates reduced botrytis rotting of lettuce leaves by more than 50% in the primary screen. Following a secondary screen against B. cinerea, the lead 50 isolates were also tested for suppression of R. solani infection. Four isolates significantly reduced both botrytis and rhizoctonia leaf rotting. Eleven and five isolates gave control of botrytis and rhizoctonia, respectively, equal to that given by the standard fungicides Rovral WP (iprodione) and Basilex (tolclofos methyl). The two most effective isolates against B. cinerea and R. solani were both identified as Bacillus subtilis. Use of soil amendments did not increase the proportion of efficacious isolates recovered. Effective isolates were originally recovered from roots of oilseed rape and lettuce leaves. In general, it was found that bacteria which controlled one disease effectively did not control the second disease nearly as well. The bioassay protocols developed in this study were used successfully in screening a large number of bacterial isolates in a short time.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

In vitro and in silico antifungal efficacy of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani

We have investigated in vitro antifungal efficiency of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani (R. solani) plant pathogenic fungi. NCNH with size of 50–60 nm and concentrations of 10, 50, 100, and 150 μg mL^?1 were used. The results showed that growth of fungi in the presence of NCNH was significantly (p > .05) inhibited at 150 μg mL^?1 (85.13 ± .97) after 72 h. The results were validated through computational approaches. Molecular docking analysis of NCNH with endochitinase protein of R. solani was performed to validate the potential of antifungal activity of NCNH. Docking results showed different conformations of interaction of NCNH with endochitinase enzyme. The conformation with least binding energy ?13.54 kcal/mol was considered further. It is likely that NCNH interacts with the pathogens by mechanically wrapping, which may be one of the major toxicity actions of NCNH against R. solani. The analysis showed that NCNH might interwinds to endochitinase of R. solani leading to the deactivation of the enzyme. To best of our knowledge, this is the first report of antifungal efficacy of NCNH against R. solani and provides useful information about the application of NCNH in resisting crop disease.     

Use of Rhizobia in the Control of Root Rot Diseases of Sunflower, Okra, Soybean and Mungbean

Biocontrol potential of Rhizobium and Bradyrbizobium against soilborne root infecting fungi was tested. In vitro tests Rhizobium meliloti inhibited growth of Macrophomina phaseolina, Rhizoctonia solani and Fusarium solani while Bradyrhizobium japonicum inhibited M. phaseolina and R. solani producing zones of inhibition. In field R. meliloti, R. leguminosarum and B. japonicum used either as seed dressing or as soil drench reduced infection of M. phaseolina, R. solani and Fusarium spp., in both leguminous (soybean, mungbean) and non-leguminous (sunflower and okra) plants.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Anti-fungal properties of 43 plant species against Alternaria solani and Botrytis cinerea

Investigation of plants containing natural anti-microbial metabolites for plant protection has been identified as a desirable method of disease control. Crude methanolic extracts of 43 plant species belonging to 27 families, which most of them are medicinal plants, mostly collected from the west of Iran were screened for anti-fungal activity against two economically important phytopathogenic fungi, Alternaria solani and Botrytis cinerea during 2010–2012. Bioassay of the extracts was conducted by agar diffusion method on agar plate cultures with five replications. Among all the 43 plant methanolic extracts, mycelia growth of A. solani and B. cinerea was reduced by 28 (65%) and 30 (70%) plant extracts when compared to the control, respectively. The strongest extracts with more than 50% inhibition against A. solani were Elaeagnus angustifolia, Dodonaea viscosa, Haplophyllum perforatum and inflorescence of Allium hirtifolium, respectively. Leaves of A. hirtifolium, H. perforatum, inflorescence of A. hirtifolium and D. viscosa showed highest inhibitory effect (≥50%) against B. cinerea. Moreover, complete inhibition of leaves of A. hirtifolium against B. cinerea was due to their fungistatic activity. The results of this experiment and high number of plants with anti-fungal activity showed that the flora in the west of Iran could be regarded as a rich source of plants with anti-fungal activity. Therefore, further screening of other plant species, identifying active fractions or metabolites and in vivo application of active extracts are warranted.     

Diverse mechanisms adopted by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> PGC2 during the inhibition of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Rhizoctonia solani and Phytophthora capsici are two of the most destructive phytopathogens occurring worldwide and are only partly being managed by traditional control strategies. Fluorescent Pseudomonas isolates PGC1 and PGC2 were checked for the antifungal potential against R. solani and P. capsici. Both the isolates were screened for the ability to produce a range of antifungal compounds. The results of this study indicated the role of chitinase and β-1,3-glucanase in the inhibition of R. solani, however, antifungal metabolites of a non-enzymatic nature were responsible for inhibition of P. capsici. The study confirmed that multiple and diverse mechanisms are adopted by the same antagonist to suppress different phytopathogens, as evidenced in case of R. solani and P. capsici.     

Greek indigenous streptomycetes as biocontrol agents against the soil‐borne fungal plant pathogen Rhizoctonia solani

Aims

To examine the biocontrol potential of multiactive Greek indigenous Streptomyces isolates carrying antifungal activity against Rhizoctonia solani that causes damping‐off symptoms on beans.

Methods and Results

A total of 605 Streptomyces isolates originated from 12 diverse Greek habitats were screened for antifungal activity against R. solani DSM843. Almost one‐third of the isolates proved to be antagonistic against the fungus. From the above isolates, six were selected due to their higher antifungal activity, identified by analysing their 16S rRNA gene sequence and studied further. The obtained data showed the following: firstly, the isolates ACTA1383 and ACTA1557 exhibited the highest antagonistic activity, and therefore, they were selected for in vivo experiments using bean seeds as target; secondly, in solid and liquid culture experiments under optimum antagonistic conditions, the medium extracts from the isolates OL80, ACTA1523, ACTA1551 and ACTA1522 suppressed the growth of the fungal mycelium, while extracts from ACTA 1383 and ACTA1557 did not show any activity.

Conclusions

These results corresponded important indications for the utility of two Greek indigenous Streptomyces isolates (ACTA1557 and ACTA1383) for the protection of the bean crops from R. solani damping‐off symptoms, while four of them (isolates OL80, ACTA1523, ACTA1551 and ACTA1522) seem to be promising producers of antifungal metabolites.

Significance and Impact of the Study

This is the first study on the biocontrol of R. solani using multiactive Streptomyces isolates originated from ecophysiologically special Greek habitats. Our study provides basic information to further explore managing strategies to control this critical disease.     

Combination of Pseudomonas aeruginosa and Pochonia chlamydosporia for Control of Root-Infecting Fungi in Tomato

A plant growth‐promoting rhizobacterium, Pseudomonas aeruginosa strain IE‐6, and a fungal antagonist, Pochonia chlamydosporia, were tested for their ability to inhibit mycelial growth of root‐infecting fungi under laboratory conditions including Macrophomina phaseolina, Fusarium oxysporum, F. solani and Rhizoctonia solani. Biocontrol effectiveness of the bacterium and the fungus alone or in combination was also determined for the control of root‐infecting fungi under field conditions. In a dual‐culture plate assay, the colonies of P. chlamydosporia and P. aeruginosa met each other and no further growth of either organism occurred. Against M. phaseolina, F. solani and R. solani, an ethyl acetate extract of the culture filtrates of P. aeruginosa inhibited fungal growth greater than the hexane extract, but against F. oxysporum the hexane extract caused greater inhibition of fungal growth. By contrast, against M. phaseolina, F. oxysporum and F. solani, the hexane extract of P. chlamydosporia was more effective in the inhibition of fungal growth than the ethyl acetate fraction. Ethyl acetate extracts of P. aeruginosa at 1.0 mg/ml not only inhibited the radial colony growth of R. solani but also lysed the fungal mycelium. P. aeruginosa produced siderophores and hydrogen cyanide under laboratory conditions. Field experiments conducted in 1997 and repeated in 1998 revealed that Pochonia chlamydosporia and P. aeruginosa significantly suppressed the root‐infecting fungi M. phaseolina, F. oxysporum, F. solani and R. solani and that the combination of the two caused greater inhibition of the fungal pathogens than either alone. Application of P. chlamydosporia and P. aeruginosa as a soil drench also resulted in enhanced growth of tomato plants.     

Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5

Two antifungal aliphatic compounds, SPM5C-1 and SPM5C-2 with a lactone and ketone carbonyl unit, respectively obtained from Streptomyces sp. PM5 were evaluated under in vitro and in vivo conditions against major rice pathogens, Pyricularia oryzae and Rhizoctonia solani. These compounds were dissolved in distilled water/medium to get the required concentrations. The well diffusion bioassay indicated that the of SPM5C-1 remarkably inhibited the mycelial growth of P. oryzae and R. solani in comparison to SPM5C-2. Though SPM5C-2 showed low antifungal activity against P. oryzae, it was not active against R. solani. Further, SPM5C-1 completely inhibited the growth of P. oryzae and R. solani at concentrations of 25, 50, 75 and 100 μg/ml. Greenhouse experiments revealed that spraying of SPM5C-1 at 500 μg/ml on rice significantly decreased blast and sheath blight development by 76.1% and 82.3%, respectively, as compared to the control with a corresponding increase in rice grain yield.     

Characterization of lipopeptides from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. (IIRAC30) suppressing <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The main aim was to identify the active compound against Rhizoctonia solani produced by the cassava endophyte Paenibacillus sp. IIRAC-30. The compounds produced were extracted with ethyl acetate and purified by Sephadex column prior to analysis by Q-TOF mass spectrometry. A C₁₅-lipopeptide with an estimated molecular weight of 1036 Da and homologues were identified. The lipopeptide had a cyclic structure, which was deduced by interpreting the ESI–MS/MS spectra of main protonated homologues containing 15:0 FA, and the amino acid composition was Glu-Leu-Leu-Val-Asp-Leu-Leu. Therefore, the lipopeptides produced by isolate IIRAC-30 was characterized as a surfactin series. Thus, the main mechanism used by Paenibacillus sp. IIRAC-30 to suppress R. solani was elucidated. Furthermore, because lipopeptides active against phytopathogens generally show low toxicity to humans and the environment, the positive findings presented here suggest that the isolate IIRAC-30 could be a possible candidate for biocontrol of R. solani.     

Molecular characterization of Rhizoctonia solani isolates the incitant of soybean root rot

Rhizoctonia solani isolates used in this investigation were identified as anastomosis-4 (AG-40), collected from different localities from Assiut governorate in Egypt. Pathogenicity test of seven isolates of R. solani was evaluated on soybean Giza 111 cultivar under greenhouse conditions. All tested isolates were able to infect soybean plants causing root rot with different degrees of severities, isolate No. 1, 2 and 3 showed significantly highest root rot severity, while isolate No. 5 gave the lowest percentage of root rot rating. The sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns were used to compare three isolates of R. solani. There are no variations among R. solani isolates except a few exceptions according to their protein patterns. DNA markers obtained from all isolates showed genetic similarity among different isolates obtained from different geographical regions barring few exceptions. Correlation between DNA patterns of R. solani isolates and their virulence was detected, but no correlation with anastomosis groups (AG).