临床常见镰刀菌的鉴别

目的从分子生物学角度寻找一种快速准确鉴定临床常见镰刀菌的方法。方法将受试镰刀菌接种于PDA培养基,观察其菌落及镜下形态,在此基础上PCR扩增受试镰刀菌的rDNA ITS并测其序列,在GenBank核酸序列数据库进行同源序列搜索及分析。选择限制性内切酶Dra Ⅱ和Cfr13 Ⅰ进行RFLP。设计了茄病镰刀菌的种特异性引物Sol1、Sol2,初步验证其特异性。结果形态学鉴定结果显示,茄病镰刀菌所占比例最高,除2株串珠镰刀菌外,其余镰刀菌ITS序列分析的结果与形态学鉴定结果一致。茄病、层生和串珠镰刀菌的Dra Ⅱ、Cfr13 I酶切带形互不相同。用Sol1、Sol2扩增受试菌的rDNA ITS,只有茄病镰刀菌为阳性。结论rDNA ITS序列测定及其PCR-RFLP可用于初步鉴别几种临床常见镰刀菌,合适的种特异性引物可以初步快速鉴定茄病镰刀菌。     

Identification, Pathogenicity and Comparative Virulence of Fusarium spp. Associated with Diseased Euphorbia spp. in Europe

Fusarium spp. isolated from diseased Euphorbia spp. in Europe were assessed for pathogenicity to North American accessions of leafy spurge ( Euphorbia esula/virgata ). Of the nine strains of Fusarium spp. isolated from diseased E. stepposa or E. virgata in the Caucasus region of Russia and E. esula/virgata in southern France, all were pathogenic to leafy spurge. There were significant differences in virulence among strains. Four strains, including the two that were most virulent, were identified as F. oxysporum . Four of the five other strains were identified as F. solani and one was identified as F. proliferatum . Three of the four most virulent strains to leafy spurge were isolated from E. stepposa . The most virulent strain was associated with root damage caused by insect biological control agents, as found earlier with domestic strains of Fusarium spp. pathogenic to leafy spurge. Two strains identified as F. solani were vegetatively compatible. It was concluded that further screening of a larger set of strains of foreign Fusarium spp. under quarantine conditions in the US or in limited overseas facilities would be justified, and could yield promising biological control agents for leafy spurge.     

Genetic diversity and relationships of diploid and tetraploid cottons revealed using AFLP

Gossypium species (± 49) represent a vast resource of genetic diversity for the improvement of cultivated cotton. To determine intra- and inter-specific genetic relationships within a diverse collection of Gossypium taxa, we employed 16 AFLP primer combinations on three diploid species, Gossypium herbaceum L. (A1), Gossypium arboreum L. (A2) and Gossypium raimondii Ulbrich (D5), and 26 AD allotetraploid accessions (Gossypium barbadense L. and Gossypium hirsutum L.). A total of 1180 major AFLP bands were observed; 368 of these (31%) were polymorphic. Genetic similarities among all taxa ranged from 0.21 (between the diploid species G. arboreum and G. raimondii) up to 0.89 (within G. barbadense). Phenetic trees based on genetic similarities (UPGMA, N-J) were consistent with known taxonomic relationships. In some cases, well-supported phylogenetic relationships, as well as evidence of genetic reticulation, could also be inferred. UPGMA trees and principal coordinate analysis based on genetic similarity matrices were used to identify genetically distinct cultivars that are potentially important sources of germplasm for cotton improvement, particularly of fiber quality traits. We show that AFLP is useful for estimating genetic relationships across a wide range of taxonomic levels, and for analyzing the evolutionary and historical development of cotton cultivars at the genomic level. Received: 17 January 2000 / Accepted: 4 May 2000     

Malachite green agar,a new selective medium for Fusarium spp.

Malachite Green Agar 2.5 ppm (MGA 2.5) is a potent selective medium for isolation and enumeration of Fusarium spp. It has been tested with pure and mixed cultures as well as in naturally contaminated samples. The recoveries of Fusarium species in MGA 2.5 were the same as the recoveries obtained in Nash and Snyder medium. However, this medium is a more selective culture medium for Fusarium spp. than Nash and Snyder medium, because it does not allow the development of colonies belonging to other fungal genera. MGA 2.5 is simple to prepare and less hazardous than other Fusarium selective media containing pentachloronitrobenzene (PCNB). This revised version was published online in June 2006 with corrections to the Cover Date.     

Quantitative Fusarium spp. and Microdochium spp. PCR assays to evaluate seed treatments for the control of Fusarium seedling blight of wheat

AIMS: To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group. METHODS AND RESULTS: Primers conserved between the species within each group were used to develop competitive PCR assays and used to quantify DNA of each group in wheat seed produced from inoculated field plots. Seed was used in seed treatment efficacy field experiments and the amount of DNA of each group was determined in emerged seedlings. The performance of treatments towards each group of pathogens was evaluated by comparison of the reduction in DNA in seedlings emerged from treated seed compared with untreated seed. CONCLUSIONS: DNA from the two groups of pathogens causing Fusarium seedling blight of wheat can be quantified separately using the competitive PCR assays. These assays show improved sensitivity compared with those previously reported for the individual species and allowed the quantification of pathogen DNA in seed and seedlings. Significant reductions in pathogen DNA were evident for each seed treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of DNA for each group allows the evaluation of seed treatment performance towards the two components of Fusarium seedling blight disease complex. The approach taken and the assays developed in this study will be of use for the study of other Fusarium disease complexes and their control. Based on the results reported here on the seedling stage of crop development, further studies that examine the control of seed-borne pathogens through fungicide seed treatments throughout the growing season are warranted.     

Characterisation,differentiation and biochemical diversity of Fusarium solani and Fusarium proliferatum based on cellular fatty acid profiles

The biochemical relationships between Fusarium solani and Fusarium proliferatum isolates were investigated using fatty acid analysis. Cellular fatty acid composition showed that palmitic acid, stearic acid, oleic acid and linoleic acid were the most abundant fatty acids in these species and accounted for 93.88 and 94.02% of the fatty acid profiles in F. solani and F. proliferatum, respectively. The most predominant fatty acids were linoleic acid (37.44%) in F. solani and oleic acid (39.81%) in F. proliferatum. The fatty acid compositions of F. solani and F. proliferatum were significantly different (p?<?0.05) for most of the individual fatty acids. This study demonstrated that fatty acid profiles may be useful to characterise and differentiate F. solani and F. proliferatum isolates at the species level. Using fatty acid analysis, biochemical diversity was observed among isolates of these species. The dendrogramme revealed that F. solani and F. proliferatum formed two distinct clusters with a distance of 7.2. Isolates of each species were clustered with each other, having a Euclidean distance of 6 and 6.6 for F. solani and F. proliferatum, respectively.     

Evaluation of Streptomyces spp. and Bacillus spp. for biocontrol of Fusarium wilt in chickpea (Cicer arietinum L.)

A study was carried out to test direct and indirect antagonistic effect against Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), and plant growth-promoting (PGP) traits of bacteria isolated from rhizosphere soils of chickpea (Cicer arietinum L.). A total of 40 bacterial isolates were tested for their antagonistic activity against FOC and of which 10 were found to have strong antagonistic potential. These were found to be Streptomyces spp. (five isolates) and Bacillus spp. (five isolates) in the morphological and biochemical characterisation and 16S rDNA analysis. Under both greenhouse and wilt sick field conditions, the selected Streptomyces and Bacillus isolates reduced disease incidence and delayed expression of symptoms of disease, over the non-inoculated control. The PGP ability of the isolates such as nodule number, nodule weight, shoot weight, root weight, grain yield and stover yield were also demonstrated under greenhouse and field conditions over the non-inoculated control. Among the ten isolates, Streptomyces sp. AC-19 and Bacillus sp. BS-20 were found to have more potential for biocontrol of FOC and PGP in chickpea. This investigation indicates that the selected Streptomyces and Bacillus isolates have the potential to control Fusarium wilt disease and to promote plant growth in chickpea.     

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。     

Antagonistic activity and hyphal interactions of Trichoderma spp. against Fusarium proliferatum and F. oxysporum in vitro

Trichoderma is a well-known antagonist against soilborne plant pathogens. However, the species and even various isolates have different biocontrol potential. To evaluate the antagonistic activities of Trichoderma harzianum, T. harzianum strain T100 (T100), T. viride and T. haematum against Fusarium oxysporum and F. proliferatum, we used dual culture and productions of volatile and non-volatile metabolites in three different phases in vitro. An analysis of the data in dual culture tests represented T. viride, T. haematum and T100 as effective antagonists of Fusarium while T100 was the only fungus being able to lyse the confronting mycelia. Similar results were obtained in the volatile metabolites tests also. In contrast with the two previous tests, the non-volatile metabolites produced by T. harzianum inhibited Fusarium mycelial growth the most, and T100 acted moderately. It was also clearly showed that the antagonistic effect of Trichoderma spp. was more on F. proliferatum than on F. oxysporum. Finally, because Trichoderma spp. was most effective in the second phase, we recommend to use T100 against F. proliferatum at the initial stages of infection as its mycoparasitism on F. oxysporum was observed microscopically through forming apressoria structures without any coiling around the pathogen.     

Aggressiveness spectrum and genetic variability of Fusarium spp. on rice and wheat varieties

Abstract

A total of 106 Fusarium spp. were isolated from infected roots and soil samples of wheat and rice. Of the 106 isolates, 32 from wheat, and 74 from rice, were isolated. Six Fusarium spp. (F. oxysporum, F. moniliforme, F. poae, F. graminearum, F. tricinctum and F. equiseti) were identified at specie level. In aggressiveness tests Fusarium spp. root rot causing fungi were screened out into different aggressiveness classes according to disease severity scales. The aggressiveness of Fusarium spp. was studied on wheat varieties (Inqalab-91 and chakwal-86) and on rice varieties (Basmati-385 and IRRI-6) under controlled conditions. The overall total number of aggressive isolates was higher in rice than in wheat. However, the percentage of severely aggressive isolates was high in wheat, whereas the percentage of moderately and slightly aggressiveness isolates was high in rice. In rice, five isolates were non-aggressive and on wheat 17 were non-aggressive. Random Amplified Polymorphism DNAs (RAPDs) were used to study the polymorphism and genetic variations within the population of Fusarium spp. that established to study correlation between taxonomical and genetical characters of fungi. Five random primers were used P1 (5′-AGGAGGACCC-3′), P2 (5′-ACGAGGGACT-3′), PE7 (5′-AGATGCAGCC-3′), P14 (5′-CCACAGCACG-3′) and PE20 (5′-AACGGTGACC-3′). Each of the 10-mer primers produced results based on the respective banding patterns they generated in present investigations. Primers distinguished the F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti. All the tested primers yielded amplification products, and that were reproducible. Although there was some intraspecific variation with primers, some strains were similar and some were different in banding pattern. In F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti were seen clustered close to one another but each primer separated them unambiguously. All primer (P1, P2, P14, PE7 and PE20) combination produced 62 bands. All primers have shown interspecific and intraspecific variations in banding patterns.     

Suppression of Fusarium Colonization of Cotton Roots and Fusarium Wilt by Seed Treatments with Gliocladium virens and Bacillus subtilis

In a growth chamber at 25 C, the fungal antagonist Gliocladium virens colonized tap roots and secondary roots of cotton in non-sterile soil after seed treatment with preparations of G. virens. Colonization of tap roots by G. virens increased over time, and decreased with root depth. Seed treatments with G. virens strains G-4 and G-6 and with Bacillus subtilis strains GB03 and GB07 reduced the colonization of tap roots and secondary roots of cotton seedlings by Fusarium spp. Under greenhouse conditions, the same seed treatments suppressed the incidence and severity of fusarium wilt of cotton in soil infested with Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita. Gliotoxin, produced by 'Q-group' strains of G. virens, inhibited F. oxysporum f. sp. vasinfectum in vitro. The seed treatments with G. virens strain G-6 and B. subtilis strains GB03 and GB07 did not affect the reproduction of root-knot nematodes. The results of this study may help to explain why the treatment of cotton seed with biocontrol agents often results in more vigorous and higher yielding plants, and indicate that there is potential for using G. virens and B. subtilis as seed treatments to control fusarium wilt of cotton.     

Effects of infection time and moisture on development of ear blight and deoxynivalenol production by Fusarium spp. in wheat

Wheat ears were inoculated with conidia of Fusarium spp. at different growth stages between ear emergence and harvest and moist conditions were maintained for up to 7 days subsequently by mist irrigation. Of the fungi tested (Fusarium culmorum, F. avenaceum, F. tricinctum, F. sporotrichioides and Microdochium nivale), only F. culmorum produced ear blight symptoms and grain samples were found subsequently to contain deoxynivalenol. Most ear infection and deoxynivalenol formation occurred following inoculation at about mid-anthesis. Small amounts of deoxynivalenol were formed and some F. culmorum was isolated even in the absence of ear blight symptoms. An overnight wet period was sufficient to initiate infection and deoxynivalenol formation but both were increased by extending the wet period up to at least 3 days. Recovery of Fusarium spp. from harvested grain was usually possible whether or not symptoms developed. F. culmorum usually persisted and often increased to moderately high levels after storage for 7 wk in a range of moisture conditions.     

陆地棉枯萎病抗性基因的等位性测定及连锁分析

1995-1996年,对我国育成的有代表性的5个抗病品种进行抗枯萎病基因的等位性测定。结果表明:在所选用的5个抗病品种中至少存在两个不同的抗病基因(暂定名为Fwl和Fw2)。连锁分析显示:Fwl与T586的8个标志性状间、Fw2与T582、T586的13个标志性状间无连锁关系。 Abstract:Allelism in vestigation of genes resistante to Fusarium wilt in cotton suggested that there were 2 genes(assigned symbols Fw1 and Fw2)in 5 cultivars used.No linkage was found between Fw1 and the marker genes in T586 and between Fw2 and those marker genes in T582 and T586.     

Isolation and characterization of endophytic streptomycete antagonists of Fusarium wilt pathogen from surface-sterilized banana roots

A total of 131 endophytic actinomycete strains were successfully isolated from surface-sterilized banana roots. These isolates belonged to Streptomyces (n=99), Streptoverticillium (n=28), and Streptosporangium (n=2) spp. The remaining 2 isolates were not identified. About 18.3% of the isolates inhibited the growth of pathogenic Fusarium oxysporum f. sp. cubense on banana tissue extract medium. The most frequently isolated Streptomyces sp. strain S96 was similar to Streptomyces griseorubiginosus. About 37.5% of the S. griseorubiginosus strains were antagonistic to F. oxysporum f. sp. cubense. The antagonism of strain S96 was lost when FeCl(3) was introduced into the inhibition zone. In vivo biocontrol assays showed that the disease severity index (DSI) was significantly (P=0.05) reduced and mean fresh weight increased (P=0.001) in plantlets treated with strain S96 compared to those grown in the absence of the biocontrol strain. These findings indicate the potential of developing siderophore-producing Streptomyces endophytes for the biological control of fusarium wilt disease of banana.     

Biocontrol efficacy of Trichoderma spp. against sesame wilt caused by Fusarium oxysporum f. sp. sesami

Sesame (Sesamum indicum L.) is one of the most important oilseed crops in Egypt and worldwide. It is being infected with many pathogens, among these pathogens Fusarium oxysporum f.sp. sesami (Zap.) Cast is causing severe economic losses on sesame. In this study, antagonistic capability of 24 isolates of Trichoderma spp. was assessed in vitro against F. oxysporum f.sp. sesami. Two strains; T. harzianum (T9) and T. viride (T21) were revealed to have high antagonistic effect against F. oxysporum f.sp. sesami in vitro with inhibition percentage about 70 and 67%, respectively. These two isolates proved to have high ability to control Fusarium wilt disease under greenhouse conditions. The highest reduction in disease severity was achieved with T. viride followed by T. harzianum with reduction in disease severity about 77 and 74%, respectively. This study revealed that the time of application of bioagents is a decisive factor in determining the efficacy of Trichoderma isolates to control Fusarium wilt of sesame. It was revealed that the highest reduction in the disease severity was achieved when either Trichoderma viride or T. harzianum were applied 7 days before challenging with the F. oxysporum f.sp. sesami.     

Anatomical and biochemical aspects of interaction between roots of chickpea and Fusarium oxysporum f. sp. ciceris race 2

Roots of the susceptible “JG-62” and resistant “WR-315” chickpeas (Cicer arietinum L.) were inoculated with a conidial suspension of Fusarium oxysporum f. sp. ciceris. Anatomical and biochemical studies were carried out in a time-course manner to elucidate the infection process and plant defence reactions. Scanning electron microscope images revealed fungal colonisation in the root hair region. Early occurrence of fungal biofilms associated with the infected “JG-62” root epidermis was also visualised. After 96 h of inoculation, a gradual accumulation of polysaccharide positive deposits was observed in the xylem vessels of the infected “JG-62” roots. Fungal mycelium was observed in the vessel lumen of infected “JG-62” after 22 days of inoculation. Due to fungal invasion during this period, some of the vessels also appeared collapsed in “JG-62”, whereas vessels in “WR-315” remained intact. The host plant defence responses specifically linked to the susceptible interactions were the induction of ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase in roots and shoots.     

河北省苹果园根际土壤中疑似致病镰孢菌种类

为了解引起河北省苹果再植病害的病原菌,在河北省10个地区苹果园中采集土壤样品,在实验室进行病原菌的诱集分离培养,根据形态和分子特征对主要病原菌进行种类鉴定。结果表明,在分离得到的293株真菌中,有116株镰孢菌,为分离频率最高的真菌。在形态学鉴定的基础上,对供试镰孢菌进行了分子鉴定。在基于核糖体基因内转录间隔区（rDNA-ITS）序列与翻译延长因子1α（EF-1α）序列片段构建的系统发育树中,代表菌株分别与GenBank登记的所属菌株聚于同一群。研究结果明确了河北省苹果再植病害的疑似致病镰孢菌,包括：尖孢镰孢Fusarium oxysporum、木贼镰孢F. equiseti、锐顶镰孢F. acuminatum、层出镰孢F. proliferatum和茄腐镰孢F. solani。     

Characterization of Fusarium spp. associated with pineapple fruit rot and leaf spot in Peninsular Malaysia

Pineapple (Ananas comosus) is one the important fruit crops planted in Malaysia, and this study was conducted to determine Fusarium spp. associated with diseases of the fruit crop as Fusarium is prevalent in tropical countries. Our objective was to identify and characterize Fusarium spp. associated with pineapple fruit rot and leaf spot mainly found on the fruits and leaves in Peninsular Malaysia. Fusarium isolates (n = 108) associated with pineapple fruit rot and leaf spot were characterized by morphological, molecular and phylogenetic analyses, a mating study and pathogenicity testing. TEF‐1α sequence analysis identified Fusarium proliferatum, Fusarium verticillioides, Fusarium sacchari and Fusarium sp. Mating was successful only between tester strains of F. proliferatum and F. verticillioides. Sexual crosses with standard tester strains showed that 82 isolates of F. proliferatum produced fertile crosses with mating population D (Gibberella intermedia) and three isolates of F. verticillioides were fertile with the tester strain of mating population A (Gibberella moniliformis). All isolates were pathogenic, causing pineapple fruit rot and leaf spot, thus fulfilling Koch's postulates.     

抗油桐枯萎病木霉菌的分离鉴定及抑菌作用研究

油桐是我国重要的木本油料植物,其生产的桐油作为天然的优良干性油在工业上具有广泛用途。但由油桐专化型尖孢镰孢菌Fusarium oxysporum f. sp. fordiis（Fof-1）侵染引起的枯萎病给油桐产业造成了毁灭性灾害,目前尚无有效防治手段。众多实践证明,利用生防菌可以有效防治土传枯萎病。本研究发现,在抗病油桐根围土壤中木霉菌的相对丰度较高,并从中分离获得了16株木霉菌;通过形态学鉴定和ITS-TEF1双基因联合构建系统进化树,鉴定出4种木霉菌：拟康宁木霉Trichoderma koningiopsis（TkonT1）、螺旋木霉T. spirale（TspiT2）、深绿木霉T. atroviride（TatrT3）和哈茨木霉T. harzianum（TharT4）;通过对峙培养试验,发现木霉菌株TkonT1、TharT4和TspiT2具有较好的抑菌效果;进一步显微观察发现菌株TkonT1和TatrT3可缠绕在尖孢镰孢菌菌丝体上或穿入菌丝体内营寄生生长,吸收病菌菌丝体养分进而导致病菌菌丝体破裂和细胞原生质消解。结果表明,从抗病油桐根围土壤中获得的拮抗木霉菌株可用于油桐枯萎病的生物防治。