Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.     

Inoculum sources of Fusarium oxysporum f.sp. asparagi in asparagus production

Fusarium oxysporum f.sp. asparagi (Foa) incites crown and root rot of asparagus which causes early decline of asparagus plantings. The aim of the present study was to identify the main inoculum sources of the pathogen in the Netherlands. As has been reported for foreign seed lots, Dutch seed lots can be infested with Foa at low levels. We found that seed infestation occurs mainly during the seed harvesting process through infested soil adhering to fallen berries. Soil samples from 59 fields without a history of asparagus growing and differing in their distance from asparagus plantings were tested for infestation with Foa, using a bioassay with asparagus as a bait plant. A high correlation was found between the incidence of infestation and proximity to asparagus fields; Foa was found in 69% of the samples from fresh fields in an asparagus production centre, and in only 6% of the samples from fields at a distance of 1 km and more from asparagus fields and outside a production centre. To evaluate planting material as an inoculum source of Foa, 49 lots of one-year-old crowns from 23 nurseries were collected and rated for disease symptoms. Infestation was found to be common with only two lots free of symptomatic plants. Most of the lots had more than 75% of symptomatic plants. Although most of the plants were infested, they showed only slight root rot symptoms. The procedure for production of Foa-free planting material is discussed. Persistence and infestation of asparagus root residues in former asparagus fields was assessed by retrieving the residues from eight former asparagus fields with an asparagus-free period of one to 25 years, and three fields with a standing asparagus crop. Even after an asparagus-free period of 25 yr asparagus root residues were retrieved from soil, although at low levels. Mean population densities of Fusarium spp. declined from 2 times 10⁶ to 1 times 10⁵ colony forming units g^_1 air-dry root tissue during the first 10 years and were still > 10⁴ c.f.u. g“¹ air-dry root tissue 20 to 25 yr after asparagus produced was stopped. The population was dominated by F. oxysporum. Eighty-three of the 112 isolates (74%) of F. oxysporum belonged to the forma specialis asparagi. The proportion of Foa in the population did not decrease in time. It was concluded that persistence of Foa in asparagus root residues is a major reason for its long-term survival.     

Biological Control of Fusarium oxysporum f.sp. asparagi by Applying Non-pathogenic Isolates of F . oxysporum

Root rot severity of asparagus plants grown in sterilized field soil inoculated with Fusarium oxysporum f . sp . asparagi (Foa) was reduced by more than 50% when the soil was precolo nized by each of 13 non - pathogenic (np) isolates of F. oxysporum originating from asparagus roots or field soils . In a greenhouse experiment , application of six np isolates to naturally infested field soil was followed by a 23 - 49% decrease of disease severity , depending on the isolate . One of them , Fo47 originating from Fusarium suppressive soil in France , was applied to field plots infested with Foa . Foa root rot was not suppressed in asparagus plants grown for 1 year in these plots . Pathogenic and np isolates extensively colonized the root surface and isolates of both types infected the roots of asparagus plants grown in sterilized field soil , with significant differences among the np isolates . Inoculation of sterilized field soil with np isolates reduced germination of Foa chlamydospores by 43 - 64% depending on the isolate used . It is concluded that np isolates of F. oxysporum can suppress asparagus root rot caused by Foa in naturally infested field soil . The differences for root colonization capacity among the np isolates imply that selection for this trait might reveal isolates that perform better under field conditions .     

Negative feedback on a perennial crop: Fusarium crown and root rot of asparagus is related to changes in soil microbial community structure

The dynamic equilibrium of an ecosystem is driven by mutual feedback interactions between plants and soil microorganisms. Asparagus exerts a particularly strong influence on its soil environment through abundant production of persistent phenolic acids, which impact selectively soil microorganisms and may be involved in Fusarium crown and root rot (FCRR) of asparagus. In a survey of 50 asparagus plantations of the province of Québec, we found that FCRR was associated with a profound cultivar-specific, reorganization of the soil microbial community, as revealed by phospholipid fatty acid (PLFA) profiling. According to PLFA indicators, microbial biodiversity as well as bacterial and fungal abundance dropped sharply with the onset of FCRR in fields planted with the cultivar Guelph Millenium. This drop was followed by a similar drop in the arbuscular mycorrhizal population. Biodiversity and microbial population size then increased to finally reach a new equilibrium. Discriminant analysis of PLFA profiles obtained from soil samples also indicated a shift in soil microbial community structure associated with FCRR development in fields planted with the cultivar Jersey Giant. Different soil biological conditions, as indicated by microbial biomass C and N and soil enzyme activities, were associated with different cultivars. Preceding crop, manure application, geographical location and tillage depth also influenced the structure of soil microbial communities in asparagus plantations, as determined by PLFA profiling. If higher FCRR incidence is a consequence of the soil microbial community reorganization, means to reduce FCRR incidence in asparagus plantations may be found among practices such as soil organic fertilization, soil tillage and intercropping strategies that would dilute the negative influence of asparagus on the soil microbial community. Finally, FCRR outbreaks were generally promoted by a previous crop of maize. It seems that maize and asparagus host a F. proliferatum teleomorph (Gibberella fujikoroi) of the same mating type.     

A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus

In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples.     

小麦种质对茎基腐病抗性评价及优异种质筛选

小麦茎基腐病是由镰孢菌侵染引起的一种世界性土传病害,近年来已严重威胁到我国小麦的安全生产。为筛选具有茎基腐病抗性的小麦种质资源,本研究采用孢子悬浮液浸种法,分别以国外抗病材料Sunco和中国品种陕253为抗病和感病对照,对670份我国小麦品种(系)进行了茎基腐病温室苗期抗性鉴定。结果发现,我国供试品种(系)感病材料(病情指数>30)所占比例达到84%,且包含多个近年来小麦生产中的主推品种,表明我国小麦品种总体抗性水平低是导致茎基腐病近年来发病频率与程度不断增加的重要原因之一。经多轮筛选,发掘获得15份抗病表现稳定、抗性水平与抗病对照Sunco相仿的材料。15份材料平均病情指数在10.9~19.4之间,其株高、抽穗期等农艺性状表现出较为丰富的变异,为我国小麦抗茎基腐病品种选育和抗性遗传研究提供了种质资源。     

Patterns of Fusarium community structure and abundance in relation to spatial, abiotic and biotic factors in soil

Members of the Fusarium genus are important components of many plant–soil systems worldwide and are responsible for many crop diseases. Knowledge of the relative influence of biotic and abiotic factors on this genus is therefore of broad economic and ecological importance. In order to address this issue, we examined Fusarium communities in soils nearby apparently healthy and symptomatic asparagus plants in 50 fields scattered in four agricultural regions of Québec, Canada. Fusarium community structure and abundance were assessed using genus-specific PCR-denaturing gradient gel electrophoresis and CFU counts, respectively. Multivariate statistical analyses were used to detect community patterns related to spatial, abiotic and biotic factors. Results suggested that Fusarium community structure (i.e. the presence and absence of the different Fusarium sequence variants in the samples) in soil is mainly related to biotic factors (arbuscular mycorrhizal fungi and bacterial community structure), whereas Fusarium abundance is more closely related to abiotic factors (mainly clay, organic matter, NH₄, Na and Cu). Some degree of influence of spatial patterns was also observed on both Fusarium community structure and abundance with, for instance, a large regional variation in Fusarium community structure. However, Fusarium community structure was not directly related to the disease status of nearby asparagus plants.     

Alternative Hosts for Fusarium spp. Causing Crown and Root Rot of Asparagus in Spain

Asparagus crown and root rot caused by Fusarium oxysporum f.sp. asparagi (Foa), F. proliferatum (Fp) and F. solani (Fs) result in early decline and loss of crop production. The role of several crop species on the survival of the Fusarium spp. was investigated. The root symptoms and plant weight of seven crop species were evaluated after inoculation with each of the three Fusarium spp. The number of colony‐forming units of the Fusarium spp. from root tissues was also determined. Garlic was shown to be a symptomatic host for Foa, Fp and Fs; Fs was also pathogenic to onion. Root colonization of garlic, onion, maize, wheat, potato and sunflower suggested that they are reservoirs of Foa, Fp and Fs from asparagus and demonstrated the importance of crop rotation on the development of this asparagus disease.     

甘肃大葱贮藏期镰孢菌腐烂病病原鉴定

【目的】大葱在贮藏期频繁发生镰孢菌腐烂病,损失严重。明确该病害病原种类对病害防治具有重要意义。【方法】利用组织分离法对采集自甘肃省兰州市(区)蔬菜市场的16份大葱贮藏期镰孢菌腐烂病病样进行病原物的分离、纯化培养,经单孢分离后根据形态学特征,再结合r DNA-ITS、EF-1a(tef)基因序列分析的方法进行鉴定。【结果】共分离得到80株镰孢菌,经鉴定分属3个种,即层出镰孢菌(Fusarium proliferatum)、尖孢镰孢菌(F.oxysporum)和燕麦镰孢菌(F.avenaceum),其中层出镰孢菌为大葱镰孢菌腐烂病的优势致病菌,分离频率为52.50%。对兰州白葱不同部位进行致病性测定,结果表明层出镰孢菌对大葱鳞茎的致病力最强,而燕麦镰孢菌对大葱鳞茎的致病力最弱。【结论】3种镰孢菌作为该病害的病原,属国内首次报道。     

香蕉上的镰孢菌种类及其系统发育关系(英文)

镰孢菌属真菌是香蕉上的重要病原菌,主要引起香蕉枯萎病以及香蕉冠腐病,在我国已明确引起香蕉枯萎病的病原为尖孢镰孢古巴专化型 Fusarium oxysporum f. sp. cubense(FOC)1号和4号生理小种,但是引起香蕉冠腐病的镰孢菌种类还未明确。为了解香蕉上镰孢菌在种间及种内水平上的多样性,2008–2011 年间作者从华南地区不同的水果市场及香蕉果园采集香蕉样品90份,分离得到143株镰孢菌。通过形态学观察及基于 EF-1α基因的系统进化分析鉴定出10种镰孢菌,即F. oxysporum、F. solani、F. camptoceras、F. pallidoroseum、F. stiloides、F. chlamydosporum、F.verticillioides、F. proliferatum、F. concentricum、F. sacchari,以及藤仓赤霉复合种(Gibberella fujikuroi species complex,GFC)中 3 个未定名的类群。轮纹镰孢 F. concentricum 及甘蔗镰孢 F.sacchari 是香蕉果实中最常见种,前菌为我国首次报道,后菌是首次报道与香蕉有关。对从香蕉上分离的藤仓赤霉复合种(GFC)及尖孢镰孢复合种(FOSC)的EF-1α序列进行了系统发育分析,其GFC中的27个菌株组成的单系群可分为7个不同的亚群,分别为 F.verticillioides、F. proliferatum、F. concentricum、F. sacchari 以及3个没有描述过的菌系 Fusarium sp. 1、Fusarium sp.2和 Fusarium sp.3;FOSC中的50个菌株形成2大分枝共12个谱系,分离自我国华南地区的21株尖孢镰孢形成7个谱系,其中 13株已知的香蕉枯萎病病原菌分布在3个谱系中,我国大陆的香蕉枯萎病病原菌菌株与来源于台湾地区及东南亚的菌株亲缘关系较近,FOC1号生理小种的遗传分化大于4号生理小种,FOC 1号生理小种与分离自香蕉果实上的尖孢镰孢菌的亲缘关系比与FOC 4号生理小种的亲缘关系更近。研究结果表明,我国香蕉上存在着丰富的镰孢菌种类,而且种内遗传多样性丰富。     

Reactions of six cultivars of rice (Oryza sativa L.) to three fungal diseases

Abstract

Fungal diseases are the most important threat for rice production in the world. Rice fields were visited and sampled from diseased bushes to identify fungal diseases in Kohgiluyeh and Boyer-Ahmad province, in the south-west of Iran. Fungi were isolated from diseased tissues by Agar-plate method, and purified by single-spore or hyphal-tip methods. They were identified by studying morphological characteristics. The virulence of isolated fungi was evaluated on six dominant rice cultivars, Champa of temperate region, Champa of subtropical region, Gerdeh, Shamim, Fajr and Tarom, under greenhouse conditions. The experiment was set up in a factorial experiment in completely randomized design with four replicates. The incidence of three fungal diseases: brown spot caused by Curvularia australiensis and Bipolaris cookei, foot rot caused by Fusarium globosum and Fusarium sambucinum, and black root rot caused by Exserohilum pedicellatum, was proven in this study. Although, all of the tested cultivars significantly showed different reaction to these fungi, but the cultivars Champa of subtropical region and Tarom were resistant to foot rot as well as the black root rot and the cultivar Gerdeh was resistant to brown spot disease.     

Genetic Variability of Phytopathogenic Fusarium proliferatum Associated with Crown Rot in Asparagus officinalis

Fusarium proliferatum (teleomorph: Gibberella intermedia ) is a causal agent of crown rot of Asparagus officinalis and is one potential fumonisin-producing species within the genus Fusarium . It colonizes roots and crowns of asparagus plants, but could also be isolated from symptomless asparagus spears. Fusarium proliferatum isolates obtained from perennial asparagus plantings from Austria and Germany were included in a study on detectability and variability of two essential genes of the fumonisin-gene cluster. Genetic fingerprinting of 45 isolates revealed 14 different fingerprint groups, indicating genetic heterogenicity of F. proliferatum . Most isolates differentiated into three main fingerprint clusters, but no association was found between fingerprint group and origin of the isolates. By gene-specific PCR it was shown that, in 25 isolates tested, both initial genes of the fumonisin biosynthetic pathway – FUM1 , encoding a polyketide synthase and FUM8 , a gene for a putative aminoacyl transferase – were detectable. This suggests that these isolates were able to produce fumonisins and could contribute to the detected contamination in originating asparagus spears with this mycotoxin. Thus, early detection of FUM -genes in F. proliferatum -colonized asparagus may be suited to prevent uptake of fumonisin contaminated food with the human diet. Restriction fragment length polymorphism analysis (PCR-RFLP) of the amplified FUM gene fragments revealed little sequence variability, suggesting a conserved structure of these genes within this species. However, sequence analysis confirmed intraspecific nucleotide polymorphisms of these genes.     

A formulation of neem cake seeded with Bacillus sp. provides control over tomato Fusarium crown and root rot

Fusarium crown and root rot (FCRR) caused by the fungus Fusarium oxysporum f. sp. radicis-lycopersici is a damaging soil-borne disease of tomato. A plant growth rhizobacterium, Bacillus sp. strain HN09 isolated from neem tree rhizosphere soil, was shown to inhibit the growth, germination and development of normal morphology of the FCRR pathogen. A substantial level of disease control was achieved in greenhouse trials by soil supplementation with a preparation of neem cake seeded with HN09. Dry sterilisation of neem cake before fermentation gained comparable disease control effect as those in the unsterilised treatment, whereas moist sterilisation treatment decreased the effect significantly. This bioformulation also led to significantly raised activities in tomato genes encoding pathogenesis-related proteins, hence providing an effective alternative for the control of FCRR, reducing the need for chemical fungicide and fertilisers that impact the environment.     

Development of a bioassay to assess resistance to Fusarium oxysporum (Schlecht.) in asparagus (Asparagus officinalis L.)

Fusarium oxysporum is one of the major pathogens causing root and crown rot in asparagus. Breeding of cultivars resistant to F. oxysporum would be the most efficient strategy for pathogen control. In this study, a bioassay was developed for screening seedling resistance. The non‐destructive bioassay comprises inoculation with a highly aggressive F. oxysporum isolate, incubation in a climate chamber and quantification of disease symptoms by a digital image analysing system and a PTA‐ELISA. This bioassay is simple to implement and demonstrated high reproducibility. Subsequently, it was used to determine the resistance behaviour of 16 asparagus genotypes to F. oxysporum. The asparagus cultivars revealed different levels of susceptibility, whereas the wild relative A. densiflorus was confirmed to be resistant.     

石灰碳铵熏蒸与施用生物有机肥对连作黄瓜和西瓜枯萎病及生物量的影响

采用稀释涂布平板计数法,研究了石灰碳铵及碳铵熏蒸对黄瓜和西瓜连作土壤尖孢镰刀菌数量的影响,以及熏蒸后施用生物有机肥对黄瓜和西瓜枯萎病的防控效果及植株生长的影响.结果表明:与对照相比,石灰碳铵及碳铵熏蒸后,连作土壤中黄瓜尖孢镰刀菌的数量分别下降95.4%及71.4%,西瓜尖孢镰刀菌的数量分别下降87.2%及64.2%;多因素方差分析表明,熏蒸、施用有机肥及作物种类均对土壤中尖孢菌数量、枯萎病发病率、防控率及生物量有显著影响;与未熏蒸施用普通有机肥对照相比,石灰碳铵熏蒸后施用生物有机肥能显著减少后茬黄瓜或西瓜土壤中尖孢镰刀菌的数量并显著降低枯萎病发病率,防控率高达91.9%及92.5%,同时显著增加了植株的株高、茎粗、SPAD值及干质量.表明石灰碳铵熏蒸及施用生物有机肥能够降低土壤中尖孢镰刀菌数量,有效防控黄瓜和西瓜枯萎病的发生并促进其植株生长.     

Arabidopsis defense response against Fusarium oxysporum

The plant fungal pathogen Fusarium oxysporum (Fox) is the causal agent of root rot or wilt diseases in several plant species, including crops such as tomato (Solanum lycopersicum), banana (Musa sapientum) and asparagus (Asparagus officinalis). Colonization of plants by Fox leads to the necrosis of the infected tissues, a subsequent collapse of vascular vessels and decay of the plant. Plant resistance to Fox appears to be monogenic or oligogenic depending on the host. Perception of Fox by plants follows the concept of elicitor-induced immune response, which in turn activates several plant defense signaling pathways. Here, we review the Fox-derived elicitors identified so far and the interaction among the different signaling pathways mediating plant resistance to Fox.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

Changes in Communities of Fusarium and Arbuscular Mycorrhizal Fungi as Related to Different Asparagus Cultural Factors

Asparagus (Asparagus officinalis) is a high-value perennial vegetable crop that has shown a marked decline in productivity after many years of continuous harvesting. This decline is caused by an increase in both abiotic (autotoxicity, harvesting pressure) and biotic stresses [fungal infections, mainly Fusarium crown and root rot (FCRR)]. To gain insight into disease development and possible mitigation strategies, we studied the effects of harvesting, time in the growing season, and field age on FCRR development, Fusarium species composition, and arbuscular mycorrhizal fungi (AMF) communities in both a controlled field experiment and an ecological survey of commercial fields. In one experiment, a 3-year-old asparagus field was subdivided into plots that were harvested or not and sampled throughout the growing season to assess short-term dominant Fusarium species shifts. In addition, diseased and healthy asparagus plants sampled from six commercial fields in the same geographical region were used to assess Fusarium and AMF communities in relation to different parameters. Fusarium and AMF communities were described by using a polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE) approach, and results were analyzed by mainly correspondence analysis and canonical correspondence analysis. Results showed that dominant Fusarium taxa assemblages changed throughout the growing season. Harvested plots had significantly more FCRR symptomatic plants at the end of the growing season, but this effect was not related with any trend in Fusarium community structure. Sampling site and plant age significantly influenced AMF community structure, whereas only sampling site consistently influenced the Fusarium community. Diseased and healthy plants harbored similar Fusarium and AMF communities. Shifts in Fusarium community might not be responsible for different disease incidence because they are ubiquitous regardless of plant health status or harvesting regime. The different incidence noted might rather be related to plant physiology, antagonist microbial communities, or soil parameters.     

Fitness of three Fusarium pathogens of wheat

Crown rot and head blight of wheat are caused by the same Fusarium species. To better understand their biology, this study has compared 30 isolates of the three dominant species using 13 pathogenic and saprophytic fitness measures including aggressiveness for the two diseases, saprophytic growth and fecundity and deoxynivalenol (DON) production from saprophytic colonization of grain and straw. Pathogenic fitness was generally linked to DON production in infected tissue. The superior crown rot fitness of Fusarium pseudograminearum was linked to high DON production in the stem base tissue, while Fusarium culmorum and Fusarium graminearum had superior head blight fitness with high DON production in grains. Within each species, some isolates had similar aggressiveness for both diseases but differed in DON production in infected tissue to indicate that more than one mechanism controlled aggressiveness. All three species produced more DON when infecting living host tissue compared with saprophytic colonization of grain or straw, but there were significant links between these saprophytic fitness components and aggressiveness. As necrotrophic pathogens spend a part of their life cycle on dead organic matter, saprophytic fitness is an important component of their overall fitness. Any management strategy must target weaknesses in both pathogenic fitness and saprophytic fitness.