生防菌根系定殖竞争作用对西瓜枯萎病发病机理的影响

【目的】西瓜枯萎病是由西瓜专化型尖孢镰刀菌(Fusarium oxysporum f.sp.niveum)引起的一种常见的毁灭性土传病害,对镰刀菌同属非致病性菌株与致病性菌株存在的竞争作用进行研究,有助于获得新的具有生防效果的菌株,从而拓宽西瓜枯萎病生物防治的手段。【方法】利用选择性培养基和稀释平板计数法对温室盆栽试验中西瓜根际和非根际土壤及植物组织中非致病性轮枝镰刀菌菌株(Fusarium verticillioides XA)与致病性尖孢镰刀菌(Fusarium oxysporum LD)进行计数,确定其在西瓜植株根际和组织中的定殖情况。【结果】将从田间西瓜枯萎病发病植株根部分离获得的菌株XA和LD接入健康土壤中,接种菌株XA既不会引起西瓜枯萎病发病症状,也不会影响西瓜植株生物量,但接种菌株LD导致严重发病症状。与单接种LD处理相比较,双接种(XA+LD)处理地上部鲜重和地上部干重都分别增加了151.2%和110%。XA菌株能成功定殖于西瓜根系,但在茎基部没有检测到。在接种菌株LD的处理中植物组织和土壤中致病性镰刀菌的数量达到(1.58 4.85)×104CFU/g。与单接种LD处理相比,双接种菌株XA和LD处理植物茎基部、根系、根际土壤和土体土壤致病性镰刀菌的数量分别下降63.3%、66.1%、3.3%和24.4%,根系、根际土壤和土体土壤非致病性镰刀菌的数量增加到(0.35 3.84)×104CFU/g;双接种处理对西瓜枯萎病的防效达57.8%。【结论】非致病性轮枝镰刀菌菌株XA可有效降低致病性尖孢镰刀菌LD对西瓜植株的定殖侵染能力,对西瓜枯萎病具有一定的生防效果。     

Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds

Chitin, a beta-1,4-linked polysaccharide of N-acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N-acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin alpha-tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild-type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.     

The tomato xylem sap protein XSP10 is required for full susceptibility to Fusarium wilt disease

XSP10 is an abundant 10 kDa protein found in the xylem sap of tomato. The protein displays structural similarity to plant lipid transfer proteins (LTPs). LTPs are involved in various physiological processes, including disease resistance, and some are able to bind and transfer diverse lipid molecules. XSP10 abundance in xylem sap declines upon infection with Fusarium oxysporum f. sp. lycopersici (Fol), implying involvement of XSP10 in the plant-pathogen interaction. Here, the biochemical characterization of XSP10 with respect to fatty acid-binding properties is reported; a weak but significant binding to saturated fatty acids was found. Furthermore, XSP10-silenced tomato plants were engineered and it was found that these plants exhibited reduced disease symptom development upon infection with a virulent strain of Fol. Interestingly, the reduced symptoms observed did not correlate with an altered expression profile for known reporter genes of plant defence (PR-1 and WIPI). This work demonstrates that XSP10 has lipid-binding properties and is required for full susceptibility of tomato to Fusarium wilt.     

Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes.

The strain ANU 6277 was isolated from laterite soil and identified as Streptomyces sp. closely related to Streptomyces albidoflavus cluster by 16S rRNA analysis. The cultural, morphological and physiological characters of the strain were recorded. The strain exhibited resistance to chloramphenicol, penicillin and streptomycin. It had the ability to produce enzymes such as amylase and chitinase. A bioactive compound was isolated from the strain at stationary phase of culture and identified as 3-phenylpropionic acid (3-PPA) by FT-IR, EI-MS, 1H NMR and 13C NMR spectral studies. It exhibited antimicrobial activity against different bacteria like Bacillus cereus, B. subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, P. flourescens, Staphylococcus aureus and some fungi including Aspergillus flavus, A. niger, Candida albicans, Fusarium oxysporum, F. udum and Penicillium citrinum. The antifungal activity of 3-PPA of the strain was evaluated in in vivo and in vitro conditions against Fusarium udum causing wilt disease in pigeon pea. The compound 3-PPA is an effective antifungal agent when compared to tricyclozole (fungicide) to control wilt caused by F. udum, but it exhibited less antifungal activity than carbendazim.     

Rhizoctonia wilt suppression of brinjal (Solanum melongena L) and plant growth activity by Bacillus BS2

An antibiotic-producing and hydrogen-cyanide-producing rhizobacteria strain Bacillus BS2 showed a wide range of antifungal activity against many Fusarium sp. and brinjal wilt disease pathogen Rhizoctonia solani. Seed bacterization with the strain BS2 promoted seed germination and plant growth in leguminous plants Phaseolus vulgaris and non-leguminous plants Solanum melongena L, Brassica oleracea var. capitata, B. oleraceae var. gongylodes and Lycopersicon esculentum Mill in terms of relative growth rate, shoot height, root length, total biomass production and total chlorophyll content of leaves. Yield of bacterized plants were increased by 10 to 49% compared to uninoculated control plants. Brinjal sapling raised through seed bacterization by the strain BS2 showed a significantly reduced wilt syndrome of brinjal caused by Rhizoctonia solani. Control of wilt disease by the bacterium was clue to the production of antibiotic-like substances, whereas plant growth-promotion was due to the activity of hydrogen cyanide. Root colonization study confirmed that the introduced bacteria colonized the roots and occupied 23-25% of total aerobic bacteria, which was confirmed using dual antibiotic (nalidixic acid and streptomycin sulphate) resistant mutant strain. The results obtained through this investigation suggested the potentiality of the strain BS2 to be used as a plant growth promoter and suppressor of wilt pathogen.     

Enhanced antifungal and insect α-amylase inhibitory activities of Alpha-TvD1, a peptide variant of Tephrosia villosa defensin (TvD1) generated through in vitro mutagenesis

TvD1 is a small, cationic, and highly stable defensin from the weedy legume, Tephrosia villosa with demonstrated in vitro antifungal activity. We show here peptide modifications in TvD1 that lead to enhanced antifungal activities. Three peptide variants, S32R, D37R, and Alpha-TvD1 (-G-M-T-R-T-) with variations in and around the β2-β3 loop region that imposes the two β-strands, β2 and β3 were generated through in vitro mutagenesis. Alpha-TvD1 exhibited enhanced antifungal activity against the fungal pathogens, Fusarium culmorum and Fusarium oxysporum with respective IC(50) values of 2.5 μM and 3.0 μM, when compared to S32R (<5.0 μM and >5.0 μM), D37R (5.5 μM and 4.5 μM), and the wild type TvD1 (6.5 μM). Because of the enhanced antifungal activity, this variant peptide was characterized further. Growth of F. culmorum in the presence of Alpha-TvD1 showed deformities in hyphal walls and nuclear damage. With respect to the plant pathogenic bacterium, Pseudomonas syringae pv. tomato strain DC3000, both Alpha-TvD1 and the wild type TvD1 showed comparable antibacterial activity. Both wild type TvD1 and Alpha-TvD1 displayed inhibitory activity against the α-amylase of the mealworm beetle, Tenebrio molitor (TMA) with the latter showing enhanced activity. The human salivary as well as barley α-amylase activities were not inhibited even at concentrations of up to 50 μM, which has been predicted to be due to differences in the pocket size and the size of the interacting loops. Present study shows that the variant Alpha-TvD1 exhibits enhanced antifungal as well as insect α-amylase inhibitory activity.     

Fungicidal activity of Ranunculus asiaticus and other weeds against Fusarium oxysporum f.sp. lycopersici

The effects of aqueous extracts of some common weed species against Fusarium oxysporum Schlecht f.sp. lycopersici (the causal agent of tomato wilt disease) were investigated under laboratory conditions. Anagallis foemina L., Cerastium dicotomum L., Falcaria vulgaris L., Ranunculus asiaticus L., Scorpiurus mur-icatus L. and Solanum nigrum L. extracts were the most toxic to the fungus. Further studies on buttercup (Ranunculus asiaticus L.) showed that fresh shoot extract of this species prevented growth of F. oxysporum when incorporated into agar medium. Extracts of different parts of the plant inhibited fungus growth and sporulation, but the fungitoxicity decreased with incubation period with only slight changes in the toxicity of fresh shoot extract. The shoot and fresh parts extracts were more toxic than root and dried tissue extracts. Addition of 0.5 ml fresh shoot or 1 ml fresh root extract to the growing medium significantly reduced fungal colony growth, and the effect was extract concentration dependent. Fresh shoot extract of R. asiaticus added to a liquid medium significantly reduced mycelial dry weight compared with the control, and incorporation of 0.1 g dried shoot or 0.2 g dried roots in the media strongly inhibited fungus growth. Results of a pot experiment showed no harmful effects of R. asiaticus extracts on tomato growth.     

Evaluation of Nostoc strain ATCC 53789 as a potential source of natural pesticides

The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.     

Some fungal endophytes from vegetable crops and their anti-oomycete activities against tomato late blight

AIMS: To isolate endophytic fungi from vegetable plants and examine their in vivo anti-oomycete activity against Phytophthora infestans in tomato plants. METHODS AND RESULTS: Endophytic fungi were isolated from surface-sterilized plant tissues and anti-oomycete activity was measured by in vivo assay using tomato seedlings. Endophytic fungi showing potent anti-oomycete activity were identified by morphological characteristics and nuclear ribosomal ITS1-5.8S-ITS2 sequence analysis. A total of 152 isolates were obtained from 66 healthy tissue samples of cucumber, red pepper, tomato, pumpkin and Chinese cabbage and the fermentation broths of 23 isolates showed potent in vivo anti-oomycete activity against tomato late blight with control values over 90%. The Fusarium oxysporum strain EF119, which was isolated from roots of red pepper, showed the most potent disease control efficacy against tomato late blight. In dual-culture tests, it inhibited the growth of Pythium ultimum, P. infestans and Phytophthora capsici. CONCLUSIONS: Among endophytic fungi isolated from healthy tissues of vegetable plants, F. oxysporum EF119 showed the most potent in vivo anti-oomycete activity against tomato late blight and in vitro anti-oomycete activity against several oomycete pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi showing anti-oomycete activity in vitro and in vivo may be used as biocontrol agents particularly of tomato late blight.     

黄瓜枯萎病拮抗放线菌的筛选、鉴定及发酵条件优化

【背景】黄瓜枯萎病是由尖孢镰刀菌(Fusarium oxysporum f. sp. cucumerinum)黄瓜专化型引起的土传真菌性病害,严重制约着黄瓜产业的发展。【目的】从河西走廊敦煌地区盐碱土壤中分离筛选出一株对黄瓜枯萎病病菌有良好拮抗效果的放线菌菌株,探究其分类地位及其最优发酵条件。【方法】采用稀释平板涂布法分离放线菌,平板对峙法、抑制菌丝生长速率法筛选拮抗菌株,通过培养特征、生理生化试验及16SrRNA基因序列分析确定其分类地位,利用单因素试验和正交试验方法确定其最优发酵配方及培养条件。【结果】菌株16-3-10鉴定为链霉菌属(Streptomyces sp.)菌株,最优发酵配方(g/L):小米10.0,乳糖20.0,蛋白胨1.0,NaCl 5.0,CaCO3 6.0,最优发酵条件:培养温度28°C,装瓶量50/250 mL,培养3 d,起始pH 10.0,抑菌率达82.50%,比优化前增加153.43%。【结论】菌株16-3-10对黄瓜枯萎病病菌具有显著的拮抗效果,有较好的应用前景。     

Effect of nitrogen nutrition on Fusarium wilt of tomato plants

The role of nitrate-nitrogen (NO₃-N) in relation to the development of tomato wilt caused by Fusarium oxysporum f. sp. lycopersici R1 was studied. Plants receiving 284 μg/ml nitrogen in the nutrient solution exhibited the same severe wilt symptoms as the control plants in soil. Disease decreased with increasing nitrogen levels (420, 630 and 1050 μg/ml). Apparently, the plants were also less susceptible to the disease when the concentration of nitrogen was 70 μg/ml, i. e. below the optimal level (284 μg/ml). Protein content in tissues of plants grown with different amounts of nitrogen was also determined. High nitrogen levels, which decreased disease severity, increased the protein content in leaf tissues. Of 17 amino acids only proline content increased with increasing nitrogen supply. High doses of NO₃–N decreased the phenol content and the activity of peroxidase in stem and leaf tissues of tomato plants. It is suggested that phenolic compounds and the activity of peroxidase are not significant in the resistance of tomato to Fusarlum wilt associated with high nitrogen supply.     

Role of ethylene in the protection of tomato plants against soil-borne fungal pathogens conferred by an endophytic Fusarium solani strain

An endophytic fungal isolate (Fs-K), identified as a Fusarium solani strain, was obtained from root tissues of tomato plants grown on a compost which suppressed soil and foliar pathogens. Strain Fs-K was able to colonize root tissues and subsequently protect plants against the root pathogen Fusarium oxysporum f.sp. radicis-lycopersici (FORL), and elicit induced systemic resistance against the tomato foliar pathogen Septoria lycopersici. Interestingly, attenuated expression of certain pathogenesis-related genes, i.e. PR5 and PR7, was detected in tomato roots inoculated with strain Fs-K compared with non-inoculated plants. The expression pattern of PR genes was either not affected or aberrant in leaves. A genetic approach, using mutant tomato plant lines, was used to determine the role of ethylene and jasmonic acid in the plant's response to infection by the soil-borne pathogen F. oxysporum f.sp. radicis-lycopersici (FORL), in the presence or absence of isolate Fs-K. Mutant tomato lines Never ripe (Nr) and epinastic (epi1), both impaired in ethylene-mediated plant responses, inoculated with FORL are not protected by isolate Fs-K, indicating that the ethylene signalling pathway is required for the mode of action used by the endophyte to confer resistance. On the contrary, def1 mutants, affected in jasmonate biosynthesis, show reduced susceptibility to FORL, in the presence Fs-K, which suggests that jasmonic acid is not essential for the mediation of biocontrol activity of isolate Fs-K.     

Tomatidine and lycotetraose, hydrolysis products of alpha-tomatine by Fusarium oxysporum tomatinase, suppress induced defense responses in tomato cells

Many fungal pathogens of tomato produce extracellular enzymes, collectively known as tomatinases, that detoxify the preformed antifungal steroidal glycoalkaloid alpha-tomatine. Tomatinase from the vascular wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici cleaves alpha-tomatine into the aglycon tomatidine (Td) and the tetrasaccharide lycotetraose (Lt). Although modes of action of alpha-tomatine have been extensively studied, those of Td and Lt are poorly understood. Here, we show that both Td and Lt inhibit the oxidative burst and hypersensitive cell death in suspension-cultured tomato cells. A tomatinase-negative F. oxysporum strain inherently non-pathogenic on tomato was able to infect tomato cuttings when either Td or Lt was present. These results suggest that tomatinase from F. oxysporum is required not only for detoxification of alpha-tomatine but also for suppression of induced defense responses of host.     

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.     

天麻抗真菌蛋白基因(gafp)转化彩色棉的研究

天麻抗真菌蛋白（gastrodia antifungal protein简称GAFP）是从我国传统中药天麻（Gastrodia elata Bl.）中分离到的一种具有广谱抗真菌活性的蛋白质,它对许多植物真菌病包括棉花枯萎病、黄萎病等的致病菌离体具有很强的抑制作用,因此,在植物抗真菌病基因工程上有很重要的应用价值。本研究通过花粉管通道法,将GAFP的基因gafp转入3个新疆彩色棉品种中,通过田间抗病筛选和分子检测,得到了高抗黄萎病的转基因植株,两株Southern杂交阳性植株LB-5-8和ZB-1-49对黄萎病表现整株免疫。RT-PCR的结果显示,LB-5-8和ZB-1-49中均有gafp的正确转录;离体的抑菌实验也表明,它们的蛋白粗提物对棉花黄萎病致病菌离体有明显的抑制,表明了gafp在转基因植株中的正确表达,翻译的产物具有活性。经过进一步选育和扩繁,发现转基因彩色棉后代具有稳定的、较强的抗黄萎病能力,本研究为通过植物抗病基因工程的方法防治棉花黄萎病提供了一条新的途径。     

抗灰葡萄孢霉菌乳酸菌的筛选

从80株乳酸菌中筛选出45株具有抗灰葡萄孢霉菌活性的乳酸菌菌株,其中10株具有较强抗灰葡萄孢霉菌活性。对这10株乳酸菌菌株的抗植物致病真菌谱进行了研究,这10株乳酸菌对番茄早疫病菌,甜瓜疫霉菌,甜瓜枯萎病菌,苹果炭疽病菌的生长均有较强的抑制作用。其中1株具有广谱抗植物致病真菌活性的乳酸菌菌株BX6-4为植物乳杆菌。经番茄离体叶片接种试验发现,植物乳杆菌BX6-4的发酵液能够在体外强烈地抑制灰葡萄孢霉菌的生长。     

Role of Bacillus amyloliquefaciens Y1 in the control of Fusarium wilt disease and growth promotion of tomato

The objective of this study was to examine the effects of Bacillus amyloliquefaciens Y1 on the control of Fusarium wilt disease and subsequent improvement in the growth of tomato plants. The Y1 strain strongly inhibited Fusarium oxysporum f. sp. lycopersici in vitro and also produced indole-3-acetic acid (IAA) in both the presence and absence of tryptophan. Over 96% of tomato seeds germinated when treated with either water, tryptone soy broth, or Y1 cultures, whereas root (5.40?cm) and shoot (5.15?cm) lengths were greatest in tomato seedlings treated with Y1 cultures that lacked tryptophan. Three experimental treatments – Black White medium (BW), BW medium with a commercial fungicide (BW?+?F), and Y1 culture inoculated in BW medium (Y1) – were applied to control Fusarium wilt disease under in vivo conditions. Application of Y1 culture and BW?+?F led to significantly lower disease incidence than did BW; moreover, shoot length and fresh and dry weight of both roots and shoots were greater in plants treated with Y1 than in plants treated with either BW or BW?+?F. A similar trend was observed for chitinase and β-1,3-glucanase activities in roots and leaves of tomato plants in all treatment groups over most of the experimental period. Finally, the presence of Y1 in the rhizospheric soils of Y1-treated plants resulted in a significant reduction in the populations of other bacteria. The results of our study demonstrated the effectiveness of Y1 not only in the control of Fusarium wilt disease but also for the enhancement of plant growth in cultivated tomato.     

Antifungal activity of essential oil and its constituents from Calocedrus macrolepis var. formosana Florin leaf against plant pathogenic fungi

Resistance to conventional fungicides causes the poor disease control of agriculture. Natural products from plants have great potential as novel fungicide sources for controlling pathogenic fungi. In this study antipathogenic activity of the leaf essential oil and its constituents from Calocedrus macrolepis var. formosana Florin were evaluated in vitro against six plant pathogenic fungi. Chemical analysis of leaf oil by GC/MS allowed identification of alpha-pinene (44.2%), limonene (21.6%), beta-myrcene (8.9%), beta-caryophyllene (8.2%), caryophyllene oxide (2.4%), alpha-cadinol (1.6%), beta-pinene (1.2%), and T-muurolol (1.1%) as main components. Sesquiterpenoid components of the oil were more effective than monoterpenoid components of the oil. In particular, T-muurolol and alpha-cadinol strongly inhibited the growth of Rhizoctonia solani and Fusarium oxysporum, with the IC(50) values < 50 microg ml(-1). These compounds also efficiently inhibited the mycelial growths of Colletotrichum gloeosporioides, P. funerea, Ganoderma australe and F. solani. These results showed that T-muurolol and alpha-cadinol possess antifungal activities against a broad spectrum of tested plant pathogenic fungi and could be used as potential antifungal agents for the control of fungal diseases in plants.     

Resveratrol lacks antifungal activity against Candida albicans

The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10-40 μg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 μg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 μg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.