Purification and characterization of an extracellular β-xylosidase from a newly isolated Fusarium verticillioides

An extracellular β-xylosidase from a newly isolated Fusarium verticillioides (NRRL 26518) was purified to homogeneity from the culture supernatant by concentration by ultrafiltration using a 10,000 cut-off membrane, ammonium sulfate precipitation, DEAE Bio-Gel A agarose column chromatography and SP-Sephadex C-50 column chromatography. The purified β-xylosidase (specific activity, 57 U/mg protein) had a molecular weight (mol. wt.) of 94,500 and an isoelectric point at pH 7.8. The optimum temperature and pH for action of the enzyme were 65°C and 4.5, respectively. It hydrolyzes xylobiose and higher xylooligosaccharides but is inactive against xylan. The purified β-xylosidase had a K _m value of 0.85 mM (p-nitrophenol-β-D-xyloside, pH 4.5, 50°C) and was competitively inhibited by xylose with a K _i value of 6 mM. It did not require any metal ion for activity and stability. Journal of Industrial Microbiology & Biotechnology (2001) 27, 241–245. Received 20 May 2001/ Accepted in revised form 06 July 2001     

Seed biopriming with novel strain of Trichoderma harzianum for the control of toxigenic Fusarium verticillioides and fumonisins in maize

Fusarium verticillioides is one of the most important fungal pathogens in maize causing both pre- and post-harvest losses and also capable of producing Fumonisins. In the present study attempts have been made for screening potential T. harzianum from native rhizosphere and to study its effect on Fusarium ear rot disease, fumonisin accumulation in different maize cultivars grown in India. Eight isolates of T. harzianum were isolated and T. harzianum isolate Th-8 exhibited better antifungal activity than carbendizim. Th-8 was formulated in different solid substrates like wheat bran, paddy husk, talcum powder and cornstarch. Maize seeds of kanchan (moderately resistant), pioneer (resistant) and sweet corn (susceptible) were selected for laboratory and field studies and these seeds were treated with a conidial suspension of T. harzianum at the rate of 1 × 10⁸ spore/ml and formulation at the rate of 10 g/kg. Treated seeds were subjected to evaluate F. verticillioides incidence, seed germination, seedling vigour and field emergence, yield, thousand seed weight and fumonisin production. It was found that the pure culture of T. harzianum was more effective in reducing the F. verticillioides and fumonisin incidence followed by Talc formulation than the carbendizim treated and untreated control. Formulations of T. harzianum were effective at reducing the F. verticillioides and Fumonisin infection and also increasing the seed germination, vigour index, field emergence, yield, and thousand seed weight in comparison with the control.     

一株产白藜芦醇虎杖内生真菌的分离和鉴定

从药食同源植物虎杖根中分离得到15株内生真菌,在马铃薯葡萄糖液体培养基(PDB)中发酵后对所有内生真菌发酵液进行HPLC检测,有5株菌的发酵液中相关成分与白藜芦醇标准品保留时间一致,确定5株菌能产生白藜芦醇,在此基础上测定了5株菌的生长曲线。其中菌株H8生长最旺盛,白藜芦醇产量最高。应用形态学及ITS序列分析方法对其进行鉴定,确定为拟轮枝镰孢菌Fusarium verticillioides。     

Variation in pathogenicity of Fusarium verticillioides and resistance of maize genotypes to Fusarium ear rot

Abstract

The ineffective control measures of pathogens is due to variability among their populations. Hence, the study of pathogenic variation of Fusarium verticillioides strains on maize genotypes. Six F. verticillioides infected maize ear were randomly obtained from three agro-ecological zones in Southwest Nigeria. Pathogenicity of F. verticillioides strains at 1.0?×?10⁶ spores/mL were examined in vivo based on the rating scale of 1–7 on maize genotypes T2L COMP.1.STR SYN-W-1, PVA SYN 8F2, and T2L COMP.4. The pathogens were inoculated to the maize genotypes on 8th week for disease severity and were all moderately susceptible, however, genotype T2L COMP.4 was the most susceptible. It was observed that, 9.4% were classified as highly virulent, 12.5% as virulent, 37.5% as moderately virulent, 21.8% as slightly virulent, and 18.8% as non-virulent. In all, Fusarium verticillioides strains displayed different degrees of virulence, however, maize genotype T2L COMP.4 was the most susceptible to ear rot.     

蛇足石杉中产石杉碱甲内生真菌的分离和鉴定

【目的】从野生蛇足石杉(Huperzia serrata)中分离筛选产石杉碱甲的内生真菌。【方法】采用薄层层析及高效液相色谱法对内生真菌代谢产物进行测定和分析以期分离获得产石杉碱甲菌株,运用形态及ITS序列分析方法对产石杉碱甲菌株进行鉴定,并利用连续传代方法考察菌株遗传稳定性。【结果】经筛选获得一株产石杉碱甲内生真菌NSH-5,经形态学鉴定及ITS序列分析鉴定为轮枝镰孢菌(Fusarium verticillioides),其石杉碱甲产量为11.76 mg/100 m L,菌株经20次连续传代后遗传稳定。【结论】NSH-5菌株为一株具有产石杉碱甲能力的轮枝镰孢菌,该菌株的发现为生物合成石杉碱甲提供了新的菌种资源。     

Species-specific PCR Assays for the Fungal Pathogens Fusarium moniliforme and Fusarium subglutinans and their Application to Diagnose Maize Ear Rot Disease

Fusarium moniliforme Sheldon (syn. F. verticillioides (Sacc.) Nirenberg) and F. subglutinans (Wollenweber & Reinking) Nelson Toussoun & Marasas comb. nov., two anamorphs of the so-called‘Gibberella fujikuroi species complex', are important maize pathogens. Together with F. proliferatum, F. culmorum, and F. graminearum (teleomorph: Gibberella zeae) they are involved in the stalk rot and ear rot disease of maize. All species produce secondary metabolites (mycotoxins) which are a potential health hazard for humans and animals that consume maize and maize products frequently. In this study the development of polymerase chain reaction (PCR) assays for an easy and sensitive identification of G. fujikuroi anamorphs in maize kernels are described. The primer pairs are based on sequences of randomly amplified polymorphic DNA (RAPD) fragments and are specific for F. moniliforme and F. subglutinans respectively. The PCR assays are independent of the high phenotypic variability of traits which may complicate classification by morphological characters. They detect approximately 100 to 200 fungal genomes in the presence of an excess of maize DNA. For the analysis of infected maize kernels a rapid and easy DNA extraction was used which does not introduce inhibitory substances into the PCR. Hence the assays enable an early identification and detection of the two pathogens in host tissue by plant breeders and plant health inspection services. The assays were successfully applied to identify field isolates from Poland and to detect the pathogens in maize ears of various hybrids in Germany.     

PCR-based assay for the rapid detection of fumonisin-producing Fusarium species in maize-based animal and poultry feeds in Karnataka,India

Abstract

One hundred and fourteen animal feedstuffs and eighty poultry feed mixtures commonly used for animal and poultry nutrition in Karnataka, India, were analysed for Fusarium contamination. The total counts of fusaria in animal feeds and poultry feed mixtures revealed a high incidence of F. verticillioides, being isolated from all positive samples. Most contaminated samples were maize pellets (71.4%), cotton seed (66.6%), maize powder (60%) and fine wheat bran (50%), respectively, while no Fusarium species was isolated from Bengal gram husk and wheat flakes. All the Fusarium species were identified by the PCR method using genus specific ITS and group specific FUM 1 primers. Of the 374 Fusarium isolates tested with ITS set of primers, all fusaria scored positive, whereas only 244 (65%) isolates tested positive with the FUM 1 set of primers. The specificity of the primers provides the basis for a simple, accurate and precise detection of Fusarium species that represents fumonisin producers, which are a considerable risk for animal, poultry and human health.     

Evaluation of maize inbred lines resistance to ear rot disease and fumonisin accumulation in Moghan

Reaction of 10 maize inbred lines against Fusarium verticillioides and fumonisin accumulation were evaluated under field conditions with three replications in Pars Abad-e-Moghan, Iran. For artificial inoculation, the inbred lines were inoculated with spore suspension in concentration of 1 × 10⁶ ml^?1 7–10 days after emergence of silk, using nail punch method. To evaluate the development of the disease, its severity percent and grain yield (g plant^?1) was determined two months after inoculation. Total fumonisin produced on maize grains were also evaluated by ELISA kits. The results showed inbred lines K19, K19/1 and K74/1 were susceptible and the rest of the inbred lines were moderately resistant to the diseases. Among moderately resistant inbred lines, A679 and K18 had lowest fumonisin accumulation. Average of the fumonisin accumulation under natural infection condition (control) was 3.37 mg kg^?1 while it was 29 mg kg^?1 under artificial infection condition, which was 760% more than control.