5-Aminolevulinic acid against strawberry Fusarium wilt:Bidirectional regulation of biocontrol agents and pathogens

Strawberry Fusarium wilt (SFW) is a systematic soil-borne disease caused by Fusarium oxysporum f.sp.fragaria (Fof),which infects the vascular bundles,blocking water and nutrient transport from roots to the aboveground.It is a severe pathogen which spreads rapidly and destroys strawberry production.Finding a way to control this disease is of great scientific value and practical importance.In this study,three fungi were isolated from the vascular tissues of sick strawberries in the field.After DNA sequencing,they were identified as Fof,Aspergillus fumigatus and Trichoderma harzianum,respectively,among which the first two are pathogens and the third is a probiotic.All fungi were controlled by thiophanate-methyl (TM),a commercial fungicide.On PDA medium,20 mg·L^(-1)5-aminolevulinic acid (ALA),a natural non-protein amino acid,promoted T.harzianum proliferation,but inhibited Fof and A.fumigatus.In confrontation test,the growth of Fof or A.fumigatus was inhibited by T.harzianum and exogenous ALA promoted T.harzianum growth but significantly inhibited the pathogen growth.When three species of fungi were separately or combinedly inoculated on healthy strawberry plants,T.harzianum promoted plant growth and development while Fof or A.fumigatus caused growth retardation,where Fof directly caused leaf yellowing and plant wilting.When the plants inoculated with different fungus were treated with ALA,the results turned out that ALA alleviated SFW symptoms by bidirectionally promoting T.harzianum proliferation and inhibiting Fof and A.fumigatus.Thus,ALA might be used in comprehensively controlling SFW in strawberry industry.

Inhibitive Activity of the Extracts of Plants in Artemisia against Fusarium oxysporum f. sp. vasinfectum and Fusarium momiliforme

[Objective]The aim was to provide theoretical basis for the control of cotton Fusarium wilt and cotton boll rot disease.[Method]The inhibitory activity of the extracts from three species of plants(Artemisia annual L.,Artemisia capillaris Thunb.,Artemisia argyi Levl.et Vant)against Fusarium oxysporum f.sp.vasinfectum and Fusarium moniliforme were studied under the condition of laboratory.[Result]The extracts of all the three plants in Artemisia showed strong antifungal activity against the tested pathogenic ...

应用巢式PCR检测黄瓜尖镰孢菌(Fusarium oxysporum f．sp．cucumbrum)

文章应用BIK2/BIK3和BIK1/BIK4两对引物,采用巢式PCR(Nested-PCR)技术对镰孢菌属26个菌株(代表了6个镰孢菌种)以及从黄瓜根际分离的20株真菌、6株细菌和7株放线菌共计59个菌株进行了扩增。结果显示,只有3个黄瓜尖镰孢菌的致病菌株能产生一条长度大约为800bp的片断。对接种黄瓜尖镰孢菌的黄瓜植株进行检测结果表明,巢式PCR能从接种5d后的黄瓜病样中特异性地检测出病原菌,而症状的出现需要10～13d时间。该项技术具有较高的灵敏度,适用于黄瓜枯萎病早期侵染检测研究。

棉花枯萎病菌(Fusarium oxysporum f.sp.Vasinfectum(ATK)Sndyder & Hansln的抑菌土初步研究

1987～1988年在河南新乡用相同接菌量种植感病品种,对棉花抗枯萎病品种86—1连作10年以上田块土样进行盆栽和田间小区试验。结果表明.枯萎病平均病指分别为29.4和4.4,与对照果园土比较抑菌效果分别为39.9％和85.8％。上述田块中86—1棉株根围5cm内土样,盆栽枯萎病平均病指19.5,抑菌效果60.5％。连作10年以上枯萎病圃土样,病指分别为27.2和6.8,抑菌效果为43.9％和76.2％。1990～1991年在辽宁省经作所试验结果,连作10年以上枯萎病圃田间抑菌效果56.2％,连作5年病圃抑菌效果17.6％。上述结果证明,枯萎病圃衰退的主要原因是棉花枯萎病菌抑菌土的产生和存在。土样内枯萎菌菌量测定结果表明,3个抑菌土土样内菌量比果园导菌土减少48.6％～59.2％。抑菌土经高压灭菌后抑菌效果完全消失,初步证明抑菌因子可能以生物因子为主。

从土壤中分离棉枯萎病菌(Fusarium oxysporum f.sp.vasinfetum (Atk) Synder & Hansen)选择性培养基研究

本文介绍一种用于分离检测土壤中棉枯萎病菌的选择性培养基—植选2号。其成分为:KH_2PO_41g,MgSO_4·7H_2O 0.5g,K_2S_2O_50.2g,KCl0.6g,NH_4NO_3 0.5g,蛋白胨5g,山梨糖10g,蔗糖5g,琼脂20g,蒸馏水1000ml,PCNB 620mg,Oxgall1g,硫酸链霉素300mg,盐酸金霉素75mg。根据棉枯萎病在此培养基上的形态特征,能较容易地识别和检测该病菌。

Effects of Medium Nutrients on Fusarium oxysporum Schl. f.sp

[Objective] To study the effects of different culture conditions on the Fusarium oxysporurn SchL f. sp. [Method] Based on species identification of the pathogenic organism of Fusarium oxysporum Schl. f. sp, effects of different cultures and different nutrients on the mycelial growth and conidial production of Fusarium oxysporum SchL f. sp were studied. [Result] The mycelial growth and conidial pro- duction of Fusarium oxysporum SchL f. sp was different under different culture con- ditions. PDA medium was the most suitable medium for the mycelial growth and had the highest conidial production; and the mycelial grew the fastest on the medium with maltose as carbon source or peptone as nitrogen source, which also had the highest conidial production. [Conclusion] This study provided experimental basis for the study of Fusarium oxysporum SchL f. sp and also provided theoretical basis for the study and control of Fusarium oxysporum Schl. f. sp.

棉花枯萎病菌(Fusarium oxysporum f. sp. Vasinfectum)突变体的诱发与鉴定

通过对32个棉花枯萎病菌菌株进行的突变体诱发与鉴定实践及技术方法探索,进一步验证了可以采用KClO3培养基来诱发突变体,并利用各突变体对不同N源的利用能力差异而采用MM,MH,MA,MO2培养基对各突变体类型进行鉴定。实验结果显示绝大多数菌株(93.7%)在KClO3培养基上能诱发出突变体,但多数(85%)突变体类型不全,且不同菌株诱得突变体的难易不同,个别菌株很难诱发到突变体。各突变体的诱发率高低依次为nit1>nit3>nit8>nitM,与国内外相关报道结果基本一致。

The effects of Trichoderma on preventing cucumber fusarium wilt and regulating cucumber physiology

In our previous studies, we identified 3 Trichoderma strains with anti-Fusarium oxysporum activity, including T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886. Here, we evaluated the effects of these 3 Trichoderma strains on preventing cucumber fusarium wilt through pot culture and greenhouse culture experiments. All 3 Trichoderma strains demonstrated higher control effects toward cucumber fusarium wilt than previous studies, with efficacies over 78%. Additionally, inoculation with the 3 Trichoderma strains significantly promoted the quality and yield of cucumbers. Among the 3 strains, Trichoderma 866 was the most effective, with disease control efficacy of 78.64% and a cucumber yield increase of 33%. Furthermore, seedlings inoculated with Trichoderma exhibited significantly increased measures of plant height, stem diameter, leaf area, aboveground fresh weight, underground fresh weight, chlorophyll content, and nitric nitrogen content, as well as the activities of several stress-resistance enzymes, including superoxide dismutase(SOD), peroxidase(POD), catalase(CAT), polyphenol oxidase(PPO), and ascorbate oxidase(AAO). In addition, the plants inoculated with Trichoderma showed decreased cell membrane permeability and malondialdehyde(MDA) content in the leaves. Together, our results suggest that T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886 inoculations inhibit F. oxysporum infection, stimulate the metabolism in cucumbers, and enhance the activities of stress-resistance enzymes, which consequently promote the growth of cucumber plants, prevent cucumber fusarium wilt, and improve the yield and quality of cucumbers. T. harzianum is a commonly used biocontrol fungus, while few studies have focused on T. asperellum or T. koningense. In this study, strains of T. asperellum and T. pseudokoningii showed excellent plant disease prevention and growth promoting effects on cucumber, indicating that they also have great potential as biocontrol fungi.

A study on the pathogen species and physiological races of tomato Fusarium wilt in Shanxi,China

In order to clarify the main pathogens of tomato Fusarium wilt in Shanxi Province, China, morphological identification, elongation factor 1 alpha （EF-1α） sequence analysis, specific primer amplification and pathogenicity tests were applied to study the isolates which were recovered from diseased plants collected from 17 different districts of Shanxi Province. The results were as follows： 1） Through morphological and molecular identification, the following 7 species of Fusarium were identified： F. oxysporum, F. solani, F. verticillioides, F. subglutinans, F. chlamydosporum, F. sporotrichioides, and F. semitectum; 2） 56 isolates of F. oxysporum were identified using specific primer amplification, among which, 29, 5 and 6 isolates were respectively identified as F. oxysporum f. sp. lycopersici physiological race 1, race 2, and race 3; 3） pathogenicity test indicated the significant pathogenicity of F. oxysporum, F. solani, F. verticillioides, and F. subglutinans to tomato plant. Therefore, among these 4 species confirmed as pathogenic to tomato in Shanxi, the highest isolation rate （53.3％） corresponded to F. oxysporum. Three physiological species, race 1, race 2, and race 3 of F. oxysporum f. sp. lycopersici are detected in Shanxi, among which race 1 is the most widespread pathogen and is also considered as the predominant race.

Isolation and identification of Fusarium oxysporum f. sp. cubense in Fujian Province, China

Fusarium wilt, caused by Fusarium oxyporum f. sp. cubense(Foc), is the most serious disease affecting banana production.To clarify the distribution of the Foc races in Fujian Province of China, 79 soil samples were collected from four regions of Zhangzhou City, the primary banana production area in Fujian. We isolated and identified 12 Foc strains based on internal transcribed spacer(ITS) sequence analysis, PCR amplification by using Foc-specific primers and pathogenicity assays.Our analysis indicated that 11 isolates belong to Foc race 1, and 1 isolate belongs to the Foc tropical species race 4(TR4).Although TR4 has previously been reported to occur in primary banana-producing provinces, such as Hainan, Guangxi,and Guangdong of China, this is the first report of TR4 isolated from the soil in Fujian Province. Monitoring the presence of Foc, in particular, the TR4 strains in the soil, is the basic strategy to prevent and control Fusarium wilt.

Isolation and Identification of Shoot-Tip Associated Endophytic Bacteria from Banana cv. Grand Naine and Testing for Antagonistic Activity against <i>Fusarium oxysporum</i>f. sp. <i>cubense</i>

Endophytic bacteria colonizing the shoot-tips of banana cv. Grand Naine were isolated and tested for the antagonistic activity against the Panama wilt pathogen Fusarium oxysporum f. sp. cubense (Foc). Pre-isolation, the suckers were given extensive disinfection treatments and the homogenate from the excised shoot-tip portion was plated on nutrient agar (NA) and trypticase soy agar (TSA). This yielded altogether 47 isolates: 26 on NA and 21 on TSA, respectively, from the 10 suckers collected during August to February. The number of bacterial isolates obtained per sucker varied from one to 15 based on colony characteristics registering up to 10 distinct species per shoot-tip based on 16S rRNA sequence analysis. The 47 isolates belonged to 19 genera and 25 species under the phylogenetic classes of Actinobacteria, α- and γ-Proteobacteria and Firmicutes. Actinobacteria constituted the predominant phylum (55% isolates) with the constituent genera of Arthrobacter, Brevibacterium, Corynebacterium, Curtobacterium, Kocuria, Kytococcus, Micrococcus, Naumanella, Rothia and Tessaracoccus spp. and an unidentified isolate belonging to the family Frankiaceae. Proteobacteria constituted the second major phylum (Brevundimonas, Enterobacter, Klebsiella, Pseudomonas, Serratia and Sphingomonas spp.) followed by Firmicutes (Bacillus and Staphylococcus spp.). Antagonistic activity of the endophytes against Foc was tested through agar plate assays (pit and spot applications on fungal lawn) employing potato dextrose agar and NA. Endophytic Pseudomonas aeruginosa (isolate GNS.13.2a) which was associated with a single sucker showed significant growth inhibition effect on Foc while Klebsiella variicola (GNS.13.3a) and Enterobacter cloacae (GNS13.4a) exhibited moderate inhibition. The study brings out considerable sucker to sucker variation in the associated cultivable endophytic bacteria in “Grand Naine” banana and identifies a few bacterial endophytes with biocontrol potential against the devastating Foc pathogen.

DNA methylation patterns of banana leaves in response to Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.

Biological Characteristics of Fusarium oxysporum f. sp.niveum Isolated from Watermelon Cultivated in Plastic Greenhouse and Indoor Toxicity Determination of Different Fungicides in Hainan Province of China

[ Objective] The paper was to study biological characteristics of Fusarium oxysporum f. sp. niveum in watermelon cultivated in plastic greenhouse in Hainan Province of China and to determine indoor toxicity of fungicides. [ Method] Effects of various factors on mycelial growth and spore germination were stud- ied, and the lethal temperature of spores and indoor toxicity of fungicides were also determined. [Result] The fungal mycelium had a better growth rate on water- melon juice medium than the others. The optimum temperature for mycelial growth and spore germination were 28 ℃ and 30 ℃, and the optimum pH values were 7 - 8 and 7 - 9, respectively. Light-dark alternation was conducive for mycelial growth. The lethal temperature of spore was 60 ℃ for 5 min. The fungal mycelium grew better while taking fructose and glucose as carbon sources and yeast extract and peptone as nitrogen sources. Toxicity determination showed that 50% pro- chloraz-manganese chloride complex WP had the greatest inhibition effect on mycelial growth, with the ECso value of 0.730 9 μg/mL; followed by 32.5% benzoic azoxystrobin SC, 30% benzoylate· propieanazol EC, 10% difenoconazole WG, 25% bromothalonil · carbendazim WP and 25% bromothalonil WP, with the ECso values from 1.884 7μg/mL to 8. 161 0μg/mL. [Conclusion] The study provided basic data for field control against F. oxysporum f. sp. niveum on watermelon cultivated in plastic greenhouse in Hainan Province.

Tolerance of New Introgressive Hybrid and Backcross Forms Pathogenic Micromitisms (<i>Verticillium dahliae</i>Kleb and Fusarium oxysporum f.sp. vasinfectum)

The article is based on the use of experimental polyploidy method, with the introduction of new introgressive hybrid forms combining several species genotype with pathogenic Verticillium dahliae Kleb. and the effects of mycotoxins separated from the Fusarium oxysporum f.sp. vasinfectum micromicette on the yield of plant seeds. New artificial complex hypertension forms based on experimental polyploidy Verticillium dahliae Kleb. and Fusarium oxysporum f.sp. vasinfectum combine the potential of resistance to mycotoxins separated from microcrystals, making a tremendous contribution to the selection of new varieties and to the effectiveness of selection as a result of the use of genetic selective research as genetic-selective genetic-selector studies.

Can Changes in Soil Properties in Organic Banana Production Suppress Fusarium Wilt?

Organic agriculture promotes disease suppression through healthy soils by increasing biological activity and diversity through the application of organic fertilizers and increasing organic inputs. Fusarium wilt of bananas (Fusarium oxysporum f. sp. Fcubense) (Foc), also known as Panama disease, has been a devastating disease throughout the world. So far, no fungicides or cultural measures have been found that control Foc sufficiently. The aim of this research was to assess whether organic-based farming systems were more resilient than inorganic farming systems to soil borne diseases, in particular Fusarium wilts. A survey was conducted comparing five organic and five conventional banana plantations at paired sites in north Queensland, Australia. Soil samples were collected and analysed for chemical, physical and biological soil health indicators. Disease development of F. oxysporum f. sp. lycopersici in tomatoes and Foc in bananas were studied in pot trials to pursue clues for identifying Fusarium suppressive soil traits. Organic soils from the survey showed higher microbial activity and lower disease symptom expression (both with tomatoes and bananas) than conventional soils. In the survey, nematode diversity and soil sulphate content were recurring indicators in all experiments showing close correlations to pathogen growth, disease expression and plant health. Organic soils were lower in plant-parasitic nematodes and sulphate sulphur levels and higher in nematode diversity, labile soil C and microbial indicators. Soil conduciveness or suppression of Foc appeared to be largely governed by competition for carbon. Measurement of soil microbial enzyme activity, nematode community structure and diversity and possibly sulphate sulphur seem to provide a relatively reliable indicator for general disease suppression. Differences between organic and conventional agriculture cannot be related to single management practices, but may be linked to synergies among system components.

Construction of PEG-mediated Genetic Transformation and Gene Knockout System in Fusarium oxysporum f. sp.cubense Tropic Race 4

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4(Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4, the young mycelia(66.7 mg/ml) of wild-type strain of Foc TR4(WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplast formation, which consisted of 25 mg/ml driselase, 0.4 mg/ml chitinase, 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×10~7 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per microgram of DNA were obtained. AmCyan, RFP and YFP genes were stably transferred into the WT-Foc TR4, separately, using the protoplast transformation system. The gene FoOCH1 encoding α-1, 6-mannosyltransferase in the WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plant-pathogen interactions.

Characterization of Fusarium Oxysporum Isolates Obtained from Wax Gourd and Chieh-qua in China by Pathogenicity, RAMs and Sequence Analysis of the rDNA Internal Transcribed Spacers (ITS1 and ITS2)

Wax gourd (Benincasa hispida Thumb. Cogn) is called white gourd, winter melon, Chinese preserving melon, Chinese squash, and don kwa. It has been cultivated in China for over 2 300 years. It probably

Biochemical and Physiological Changes of Three Watermelon Cultivars Infested with Fusarium oxysporum f.sp.niveum

The dynamic changes in membrane permeability, activities of disease-related enzymes, and contents of pathogenesis-relevant chemical compounds and photosynthetic pigments in root cells of three watermelon cultivars were studied after inoculation with Fusarium oxysporum f. sp. niveum at seedling stage. The results showed that the capacity of self-regulating and returning to normal status of cultivar Kelunsheng (resistant) was greater than that of a susceptible cultivar, Zaohua, in terms of malonaldehyde (MDA) content, relative conductivity, and activity of superoxide dismutase (SOD) and catalase (CAT). The resistant cultivar maintained a higher activity of dehydrogenase, higher content of vitamin C(Vc) , and relatively lower content of soluble sugar than the susceptible cultivar. The content of soluble protein was higher in the resistant cultivar than that in the susceptible one at day 1 after inoculation. The capacities of the resistant cultivar to inhibit chlorophyll deterioration and maintain a higher carotenoid content were significantly stronger than those of the susceptible cultivar.

Genetic analysis of the maximum germination distance of Striga under Fusarium oxysporum f.sp.strigae biocontrol in sorghum

Maximum germination distance（MGD） is an important component of Striga resistance in sorghum. The objective of this study was to determine gene action influencing MGD of Striga hermonthica and Striga asiatica among selected sorghum lines treated with a biocontrol agent, Fusarium oxysporum f. sp. strigae（FOS） for effective breeding with Striga resistance, and FOS compatibility. Twelve sorghum genotypes were selected based on their Striga resistance, FOS compatibility, and superior agronomic performance. Selected genotypes were crossed using a bi-parental mating design to generate six families for genetic analysis. Agar-gel assays were used to determine low haustorium initiation factor（LHF） using the 12 parental lines, their F_1 progenies, backcross derivatives, and F_2 segregants in two sets. One set had S. hermonthica seed and the other one had S. asiatica seed. Both were treated with and without FOS. Genotypes were evaluated using a split-plot design with three replications and MGD data were recorded followed by generation mean analysis. FOS reduced MGD by 1cm under both S. hermonthica and S. asiatica infestations. Additive, dominance, and epistatic gene actions were involved in the control of MGD of the two Striga species in the evaluated populations. On average, the relative contribution of additive, additive×additive and dominance×dominance genetic effects on the MGD of S. hermonthica and S. asiatica, with FOS, were 20, 33, and 36%; and 21, 32, and 35%, respectively. Breeding methods exploiting these genetic effects may provide enhanced response to selection for Striga resistance and FOS compatibility in integrated Striga management（ISM） programmes.

Interaction between Allelochemicals and <i>Fusarium</i>Root Rot in Asparagus Seedlings Cultured <i>In Vitro</i>

The interaction between Fusarium root rot and 4 allelochemicals in asparagus seedlings was estimated in vitro to clarify the relationship between biotic and abiotic factors in asparagus decline. In in vitro culture of Fusarium oxysporum f. sp. asparagi (Foa) with or without addition of 4 allelochemicals (caffeic acid, ferulic acid, quercetin, malic acid;0.01%, 0.1%, w/v) using Czapec-Dox media, Foa propagation was suppressed in all the treatments. The degree of suppression became higher in 0.1% than 0.01% among all the allelochemicals. As for the axenic culture of asparagus (Asparagus officinalis L., “Welcome”) seedlings with the 4 allelochemicals, dry weight of both shoots and roots became lower compared to control in 0.1% and 0.01% of caffeic acid, 0.1% ferulic acid, 0.01% quercetin, only dry weight of shoots decreased in 0.1% malic acid. Two weeks after Foa inoculation with Foa-cultured PDA cubes in vitro, incidence of Fusarium root rot reached 100% in most of the plots. The severity of root rot increased in 0.01% and 0.1% caffeic acid, 0.1% ferulic acid, 0.1% malic acid compared to control. From these results, the 4 allelochemicals used in this study are supposed to suppress asparagus growth, and such growth reduction might enhance the disease severity of Fusarium root rot as an indirect effect. In addition, such effect might differ with the allelochemicals and concentrations in asparagus.