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.     

Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.     

Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides

A polymerase chain reaction assay was developed for detection of Fusarium sporotrichioides, a plant pathogen in many parts of the world. Based on small nucleotide differences in ITS2 (Internal Transcribed Spacer) rDNA of our local isolate of F. sporotrichioides (Accession No. AY510069) and other isolates found in NCBI/GeneBank database, species specific primer FspITS2K was selected. Primer pair FspITS2K and P28SL amplified a fragment of 288 bp containing a portion of ITS2 and 28S rDNA of all the F. sporotrichioides isolates tested, originated from different hosts and regions of the world but did not amplify any other species of Fusarium and plant's DNA. To use the PCR assay in seed health testing, a protocol was setup for the rapid and effective preparations of fungal DNA from wheat seeds. The method developed may be useful for the rapid detection and identification of F. sporotrichioides both from culture and from plant tissue.     

应用PCR技术检测串珠镰刀菌

串珠镰刀菌(Fusarium moniliform)是一种危害严重、在世界各地广泛流行的植物病原真菌,当前对串珠镰刀菌的鉴定主要根据其菌丝体及再生菌丝的形态结构学特征及染病作物的病害症状来进行鉴定。这些鉴定方法相对简单并在很大程度上依赖于经验,受主观因素影响较大。采用串珠镰刀菌种特异性的寡聚核苷酸为引物,运用PCR技术对串珠镶刀菌进行检测是一种快速可靠的检测鉴定方法,它无需病原菌的分离培养纯化,能从感病的玉米组织中直接实现对串珠镰刀菌的快速检测。经对霉变玉米样品和玉米穗腐病组织的检测,证明该方法是一种快建、有效的方法,具有重要的实际应用价值。     

Fusarium eumartii Growth in Resistant and Susceptible Oak Species

Fusarium eumartii is a fungus associated with declining Quercus robur , in which it is found in the vessels. The response of oak species to infection is known to vary: Q. robur is susceptible , but Quercus cerris and Quercus pubescens are resistant. An experiment was carried out in 1996 and repeated in 1997, to examine how F. eumartii colonization differed in oak species that were susceptible or resistant to the fungus by counting the number of vessels with mycelium at various distances from the inoculation site in infected seedlings and by determining the amount of viable fungus in infected tissue. Infected vessels with mycelium were counted on sections (10  μ m thick) cut at 0, 2, 4, 6, 8 and 10 cm from the inoculation site on 1-year-old inoculated seedlings as well as on sections cut every 2 cm to the seedling tip. The amount of viable fungus was determined by counting the colony forming units (CFUs) in stem segments from the same seedlings. Quercus robur seedlings had the greatest number of infected vessels and the greatest number of CFUs. Forty days after inoculation, the extent of vertical fungal spread was 28.12 cm in Q. robur , 3.15 cm in Q. cerris and 3.00 cm in Q. pubescens . The greatest number of CFUs was found in Q. robur at day 5 after inoculation. Analysis of variance confirmed the results.     

中国的膨孢组镰孢菌(英文)

膨孢组镰孢菌Fusarium在自然界中广泛分布。该组包括4个种:木贼镰孢菌F.equiseti,藨草镰孢菌F.scirpi,长脚镰孢菌F.longipes和紧致镰孢菌F.compactum。这4个种均产生腹背不平行弯曲的大孢子。分离获得并描述了其中的3个种。木贼镰孢菌F.equiseti是镰孢菌中最常见的种之一,它产生典型的腹背不平行弯曲的大孢子,大孢子的顶细胞和足跟状基细胞明显伸长,菌落因缺乏红色素而呈黄褐色。藨草镰孢菌F.scirpi是比较少见的种类,它的典型特征是在典型的十字形产孢细胞上产生大量的小型分生孢子。由于它在PDA培养基上容易发生小孢子缺乏型的变异,因此,常常被错误地鉴定为木贼镰孢菌F.equiseti。长脚镰孢菌F.longipes的大孢子最容易与其他种的大孢子区分,它的顶细胞和基细胞均极度延长。当长脚镰孢菌F.longipes菌落因为变异而失去产生红色素的能力时,也容易与木贼镰孢菌F.equiseti混淆。基于该组大孢子的典型特征,作者将锐顶镰孢菌F.acuminatum排除在该组之外。     

Identification of Spanish barbel species using the RAPD technique

The RAPD-PCR technique was employed to identify three endemic Spanish species of Barbus: Barbus bocagei, B. graellsii and B. sclateri , that present very similar morphologies. Using seven primers, six diagnostic bands were found in B. bocagei , 11 in B. graellsii and nine in B. sclateri . Cluster analysis of the genetic similarity values obtained from RAPD data indicated that the species B. bocagei and B. graellsii are more related to each other than to B. sclateri .     

Fusarium Species from the Fusarium fujikuroi Species Complex Involved in Mixed Infections of Maize in Northern Sinaloa,Mexico

Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) are associated with maize in northern Mexico and cause Fusarium ear and root rot. In order to assess the diversity of FFSC fungal species involved in this destructive disease in Sinaloa, Mexico, a collection of 108 fungal isolates was obtained from maize plants in 2007–2011. DNA sequence analysis of the calmodulin and elongation factor 1α genes identified four species: Fusarium verticillioides, F. nygamai, F. andiyazi and F. thapsinum (comprising 79, 23, 4 and 2 isolates, respectively). Differential distribution of Fusarium species in maize organs was observed, that is F. verticillioides was the most frequently isolated species from maize seeds, while F. nygamai predominated on maize roots. Mixed infections with F. verticillioides/F. thapsinum and F. verticillioides/F. nygamai were detected in maize seeds and roots, respectively. Pathogenicity assay demonstrated the ability of the four species to infect maize seedlings and induce different levels of disease severity, reflecting variation in aggressiveness, plant height and root biomass. Isolates of F. verticillioides and F. nygamai were the most aggressive. These species were able to colonize all root tissues, from the epidermis to the vascular vessels, while infection by F. andiyazi and F. thapsinum was restricted to the epidermis and adjacent cortical cells. This is the first report of F. nygamai, F. andiyazi and F. thapsinum infecting maize in Mexico and co‐infecting with F. verticillioides. Mixed infections should be taken into consideration due to the production and/or accumulation of diverse mycotoxins in maize grain.     

腐皮镰孢菌壳聚糖酶的酶学性质研究及其在酿酒酵母工业菌株中的表达

摘要：【目的】本研究旨在了解腐皮镰孢菌（Fusarium solani）壳聚糖酶的基本酶学性质及其在壳寡糖生产中的应用,构建能高效分泌表达壳聚糖酶的酿酒酵母工业菌株。【方法】采用RT-PCR扩增腐皮镰孢菌壳聚糖酶的cDNA序列;通过组氨酸标签,纯化得到E. coli表达的重组壳聚糖酶,并进行基本酶学性质研究;以薄层层析、高效液相色谱等技术对该酶的酶解产物进行分析;通过马克斯克鲁维酵母（Kluyveromyces marxianus）菊粉酶信号肽（INU1A）实现壳聚糖酶在酿酒酵母工业菌株N-27中的分泌表     

Fusarium Species Isolated from Common Weeds in Eggplant Fields and Symptomless Hosts of Fusarium oxysporum f. sp. melongenae in Turkey

Thirteen species of weed plants were collected between May and September in 2010 and 2011 from eggplant fields representing 11 distinct locations covering a wide geographical area of Turkey. Weeds are potential hosts of many plant pathogens and may not exhibit disease symptoms when colonized. Fusarium spp. were isolated from five monocotyledonous species and eight dicotyledonous species. A total of 212 isolates recovered from weeds were assigned to eight Fusarium species on the basis of morphological characteristics. F. oxysporum was the most frequently isolated species (29.7%), followed by F. solani (19.8%), F. graminearum (13.7%), F. verticillioides (12.7%), F.equiseti (9.9%), F. avenacearum (8.0%), F. proliferatum (3.8%) and F. subglutinans (2.4%). The F. oxysporum isolates from different weed hosts were characterized by means of pathogenicity and vegetative compatibility grouping (VCG) tests. Among these, 29 isolates were found to be pathogenic to eggplant cv. Kemer and re‐isolated as Fusarium oxysporum Schlecht. f. sp. melongenae (Fomg) as evidenced. These isolates from weed hosts were assigned to VCG 0320. This study is the first report of Fomg isolated from weeds in eggplant fields in Turkey. None of the weed species tested showed symptoms of wilting in pot experiments, and F. oxysporum was isolated with greater frequency from all inoculated weeds. The results of this study indicate that several weed plants may serve as alternative sources of inoculum for Fomg, during the growing season.     

Development of PCR assays for the detection and differentiation of Fusarium sporotrichioides and Fusarium langsethiae

Isolates of the type-A trichothecene producing Fusarium sporotrichioides and Fusarium langsethiae were grouped and differentiated in a phylogenetic tree using ITS sequence dissimilarity. An attempt was made to develop a PCR-based assay for the detection and differentiation of Fusarium sporotrichiodes from other Fusarium species using the 5'-region of the tri5 gene as a template. However, this assay was unable to differentiate, to a satisfactory level, between isolates of Fusarium sporotrichioides and Fusarium langsethiae, providing further genetic evidence for their close genetic relationship. A robust and repeatable PCR-assay was developed for the detection and differentiation of both species based on sequence determined from differentially amplified RAPD-PCR products. These assays were able to detect both species in samples of grain taken from the field.     

RAPD Characterization of Fusarium oxysporum Isolates Pathogenic on Argyranthemum frutescens L.

The random amplified polymorphic DNA (RAPD) technique was used to analyse total genomic DNA of 10 isolates of a new Fusarium oxysporum pathogenic on Argyranthemum frutescens (Paris daisy), by comparing them with representatives of the formae speciales basilici, chrysanthemi, cyclaminis, dianthi, gladioli, lilii, lycopersici, melonis, pisi, radicis‐lycopersici, tracheiphilum, and a non‐pathogenic isolate of F. oxysporum. A close genetic relatedness was observed among most of the new isolates from A. frutescens. These isolates also shared RAPD markers with the tested representatives of the forma specialis chrysanthemi. A single isolate among those tested from diseased A. frutescens was placed in a different cluster, which included representative isolates of forma specialis tracheiphilum. All the new isolates from A. frutescens, with the exception of the single divergent one, could be identified by their characteristic amplification profile, using selected random primers. A rapid protocol for DNA extraction directly from fungal colonies grown on Fusarium selective medium allowed the complete analysis in less than 4 h.     

Fusarium Species Associated with Mango Malformation in Peninsular Malaysia

Mango malformation has become the most important global disease on mango. Fusarium species previously associated with this disease include F. mangiferae, F. mexicanum, F. sterilihyphosum, F. proliferatum, F. subglutinans and F. tupiense. A few strains of F. proliferatum have been reported from Malaysia, but in this study, we report the results of more extensive sampling. The recovered strains were evaluated with morphology, mating tester strain cross‐fertility, amplified fragment length polymorphisms (AFLPs), and partial DNA sequences of the genes encoding translation elongation factor 1‐α (tef‐1α) and β‐tubulin (tub‐2). Amongst the 43 strains evaluated, three species were identified – F. proliferatum, F. mangiferae and F. subglutinans – with F. proliferatum being the most frequent (69%). None of the Fusarium species that appear to originate in the Americas were recovered in Malaysia, which suggests special measures may be warranted to keep these species from entering the country.     

Binding of Fusarium mycotoxins by fermentative bacteria in vitro

AIMS: Fusarium toxins can occur in conserved forages impairing farm animal performances and health. On-farm biological decontamination methods could be an alternative to traditional physico-chemical methods. In this work, the ability to remove Fusarium toxins by fermentative bacteria was evaluated in vitro. METHODS AND RESULTS: Twenty-nine strains of lactic (LAB) and propionic acid bacteria (PAB) were tested for their ability to remove deoxynivalenol (DON) and fumonisins B1 and B2 (FB1, FB2) from an acid, pH 4, medium. Mycotoxin removal was widespread for LAB, but differences among strains were large. Removal was up to 55% for DON, 82% for FB1 and 100% for FB2. Selected strains were also capable of removing up to 88% zearalenone. The PAB strains were less efficient than the LAB. Binding, not biodegradation appeared to be the mode of action, as no toxin derivatives were observed and removal was not impaired in nonviable bacteria. Binding was not affected by pH, except for fumonisins that decreased to nearly 0% at neutral pH. CONCLUSIONS: Selected fermentative bacteria are able to bind main Fusarium mycotoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The binding ability of selected strains could be used to decrease the bioavailability of toxins in contaminated silages.     

镰刀菌属一个中国新记录种

从山西省13个主要土壤类型的1 012个土壤样品中分离出镰刀菌333株,依据布斯的镰刀菌属分类系统鉴定其中1株为中国新记录种——柔毛镰刀菌(Fusarium flocciferum Corda),并对其进行描述和讨论。菌种保存于山西农业大学农学院植物病理实验室。     

Fusarium Species From Plant Debris Associated With Soils From Maize Production Areas in the Transkei Region of South Africa

Fusarium species were isolated from plant debris in soil samples collected from cultivated maize fields and from undisturbed grasslands in two areas of the Transkei region. A total of 1205 Fusarium isolates were recovered from 27 soil samples. Fifteen Fusarium species were recovered from plant debris from Bizana soils and 13 Fusarium species from plant debris from Centane soils. The two dominant Fusarium species in both areas were F. oxysporum and F. equiseti. Very few isolates of F. moniliforme and F. subglutinans were recovered, but both of these species had significantly higher relative densities in cultivated soils than in undisturbed soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence of Toxic Hexadepsipeptides in Preharvest Maize Ear Rot Infected by Fusarium poae in Poland

Twenty‐seven preharvest maize ears affected by Fusarium poae rot (disease score 36–100%) were selected in 1998 and 1999 in Poland and examined for the occurrence of toxic hexadepsipeptides: beauvericin (BEA), enniatin A, enniatin B and enniatin B₁. The identification of F. poae was confirmed by sequence analysis of variable internal transcribed spacer regions and compared with NCBI gene bank DNA sequences. Chemical analyses were performed by HPLC‐MS. In 27 ears infected by F. poae were detected: BEA (trace to 46 μg/g) in 18 samples, enniatin A (trace to 37 μg/g) in nine samples, enniatin B (trace to 47 μg/g) in 15 samples and enniatin B₁ (trace to 25 μg/g) in 12 samples. When 20 strains of F. poae isolated from these samples were cultured on rice, all produced BEA (1.9–75 μg/g), three enniatin A (1.8–2 μg/g), 12 enniatin B (1.1–5.1 μg/g) and eight enniatin B₁ (1.2–5.2 μg/g). Occurrence and quantification of enniatin A, enniatin B and enniatin B₁ and their co‐occurrence with BEA in maize kernels is reported for the first time.     

镰刀菌真菌毒素产生与调控机制研究进展

镰刀菌是一种重要的植物病原菌,给世界范围内农作物生产带来巨大破坏。除导致产量下降外,由其产生的镰刀菌真菌毒素能够污染农产品品质,给动物和人类食物安全造成严重隐患。单端孢霉烯族毒素（Trichothecenes）、伏马菌素（Fumonisin）和玉米赤霉烯酮（Zearalenone）是三种最重要的镰刀菌真菌毒素。镰刀菌真菌毒素的生物合成与生产受到体内一系列相关功能基因的调控;此外,pH值、碳氮比等环境条件也能影响真菌毒素的产量。本文简述了镰刀菌真菌毒素在产生机理、主要分类、致病性以及调控因素等方面的研究进展。     

Identification and genetic diversity of Fusarium sacchari associated with pokkah boeng disease of sugarcane in Northeast Brazil

Isolates from the Fusarium fujikuroi species complex, mainly F. sacchari, have been reported to be the causal agents of pokkah boeng in sugarcane in Brazil. However, inadequate information was available on the occurrence and genetic diversity of F. sacchari in Northeast Brazil, which is a limiting factor on management. Thus, isolates of F. subglutinans sensu lato from sugarcane plants with symptoms of pokkah boeng were evaluated using the sexual cross-fertility to determine species. All the isolates produced black perithecia when they were crossed with the test isolates of F. sacchari. Three weeks after the crossing, the formation of fertile ascospores cirri was observed. Thirty-four isolates were self-sterile hermaphrodites, while 21 were fertile only as males. Five isolates were homothallic. The effective size [N_e(f)] of the population as a function of the frequency of hermaphrodites and female sterile strains was 95.5%. The F. sacchari isolates were separated into four genetic groups independent of geographic location. The mean of polymorphism among all populations was 79%, and the average unbiased genetic diversity (uh) was considered moderate (0.31). This study in addition to confirming that F. sacchari as the main species associated with pokkah boeng in sugarcane in Northeast Brazil, reveals the relationship of mating type and genetic diversity of F. sacchari. The unrestricted gene flow between regions is probably the best explanation for the low geographic correlation. This knowledge will help in the adoption of management measures with fungicides or resistant cultivars.