Characterization of Fusarium spp. responsible for causing dry rot of potato in Great Britain

A 3-year survey was undertaken to establish the relative frequency of different Fusarium spp. present as inoculum on potato tubers collected from four regions of Great Britain. A total of 219 samples (comprising 10 950 tubers) were collected from the 2000, 2001 and 2002 crops and processed to recover dry rot-producing isolates. In total, 228 isolates of Fusarium spp. were recovered. Most (94·7%) of these isolates were attributed to one of four Fusarium species: F. coeruleum, F. avenaceum, F. culmorum and F. sambucinum (formerly F. sulphureum) . The incidence of the combined Fusarium spp. increased the further south the crops had been grown. Fusarium coeruleum was the most commonly isolated species in each survey year, comprising 37 to 52% of the total Fusarium species. Selected isolates of each species were evaluated for their ability to produce rots in potato tubers. Fusarium sambucinum was a more aggressive pathogen than the other Fusarium species in eight out of 10 cultivars. Fusarium avenaceum and F. culmorum were relatively weaker pathogens. However, these species were aggressive on some cultivars, notably Hermes. The selected isolates were also assessed for their sensitivity to the fungicides thiabendazole and imazalil. Using in vitro tests, 65% of F. sambucinum isolates were resistant to thiabendazole and 7% of F. avenaceum isolates were resistant to imazalil. Tubers treated with imazalil yielded a higher proportion of isolates of F. avenaceum than those that were untreated. Similarly, a higher proportion of F. sambucinum isolates were recovered from tubers treated with thiabendazole than from those that were not treated.     

Use of quantitative molecular diagnostic assays to investigate fusarium dry rot in potato stocks and soil

ABSTRACT Specific and sensitive quantitative diagnostics, based on real-time (TaqMan) polymerase chain reaction (PCR) and PCR enzyme-linked immunosorbent assay, were developed to detect dry-rot-causing Fusarium spp. (F. avenaceum, F. coeruleum, F. culmorum, and F. sulphureum). Each assay detected Fusarium spp. on potato seed stocks with equal efficiency. Four potato stocks, sampled over two seed generations from Scottish stores, were contaminated with F. avenaceum, F. sulphureum, F. culmorum, F. coeruleum or a combination of species, and there was a general trend towards increased Fusarium spp. contamination in the second generation of seed sampled. F. sulphureum and F. coeruleum caused significantly (P < 0.05) more disease in storage than the other species when disease-free tubers of potato cvs. Spunta and Morene were inoculated at a range of inoculum concentrations (0, 10(4), 10(5), and 10(6) conidia/ml). Increased DNA levels were correlated with increased disease severity between 8 and 12 weeks of storage. The threshold inoculum levels resulting in significant disease development on both cultivars were estimated to be 10(4) conidia/ml for F. sulphureum and 10(5) conidia/ml for F. coeruleum. To study the effect of soil infestation and harvest date on disease incidence, seed tubers of cvs. Morene and Spunta were planted in a field plot artificially infested with the four Fusarium spp. F. culmorum and F. sulphureum were detected in soil taken from these plots at harvest, and F. sulphureum DNA levels increased significantly (P < 0.05) at the final harvest. All four Fusarium spp. were detected in progeny tubers. There was a trend toward higher levels of F. culmorum detected in progeny tubers at the earliest harvest date, and higher levels of F. sulphureum at the final harvest. The use of diagnostic assays to detect fungal storage rot pathogens and implications for disease control strategies are discussed.     

Resistance to thiabendazole in isolates of Polyscytalum pustulans (skin spot) and Fusarium solani var. coeruleum (dry rot) in Scotland

Between 1988 and 1991, isolates of Polyscytalum pustulans were obtained from potato tubers grown in Scotland and tested for sensitivity to thiabendazole (TBZ) on agar. Sensitive isolates did not grow at 1 mg TBZ/1, whereas growth of resistant isolates was unaffected at 1 mg and reduced by 10–30% at 10 mg/1. Resistant isolates were present on all classes of seed potatoes, and were also recovered from the air of stores during grading. Isolates from a stock of seed tubers from Northern Ireland were resistant. All 44 isolates of Fusarium solani var. coeruleum collected in 1991 were sensitive to TBZ.     

Inoculating plant material by jet injection

Both tubers and plants of potato were successfully inoculated with Erwinia carotovora subsp. itroseptica by means of a needle-less medical jet injector. The instrument has also been used to infect potato tubers with Phorna exigua var. foveata, Fusarium solani var. coeruleum and F. sulphureum. It is suggested that high-pressure injection is a potentially useful tool for introducing pathogens into plant materials.     

Influence of light-enhanced glycoalkaloids on resistance of potato tubers to Fusarium sulphureum and Fusarium solani var. coeruleum

Increases in resistance to Fusarium sulphureum and Fusartium solani var. coeruleum were determined in seed tubers of cultivars Desiree, Pentland Hawk and Kerr's Pink with light-enhanced glycoalkaloids. Glycoalkaloid concentration in the tubers ranged from 78.0 to 885.9 mg kg⁻¹ fresh weight. Exposure of tubers to light prior to inoculation decreased fungal invasion, the greater reductions generally reflecting longer light exposure periods. In most cases, no direct relationship between resistance and glycoalkaloid concentrations was recorded for F . sulphureum. Surface infection indices and reduced depth of rot caused by F. solani var. coeruleum were closely correlated with tuber glycoalkaloid content. No significant effects on spore length and width were recorded. Exposure to light of seed tubers that are not destined for human or animal consumption could provide a pretreatment to reduce fungal invasion by F. sulphureum and F. solani var. coeruleum .     

黑龙江省马铃薯干腐病菌种类鉴定及致病性

本研究将采自黑龙江省不同地区的马铃薯干腐病病样进行分离和病原菌纯化,得到27个镰刀菌菌株,通过致病性鉴定,其中的18个菌株具有致病性。运用培养性状和形态特征综合分析的方法,对上述18个菌株进行鉴定,结果显示为6种镰刀菌,分别为拟枝孢镰孢(Fusarium sporotrioides)、茄镰孢(F.solani)、接骨木镰孢(F.sam-bucinum)、拟丝孢镰孢(F.trichothecioides)、燕麦镰孢(F.avenaceum)和茄病镰孢蓝色变种(F.solanivar.coerule-um)。同时对上述6种镰刀菌进行致病性测定,结果表明不同种类镰刀菌致病性不同,以接骨木镰孢、燕麦镰孢和拟丝孢镰孢致病力最强,拟枝孢镰孢致病力最弱。     

Pathogen resistance in aerial tubers of potato cultivars

Increases in resistance of aerial tubers (cultivars Désirée, Epicure and King Edward) against nine pathogenic potato fungi were determined in vitro using freeze-dried powder derived from subterranean and aerial tubers as a culture medium, and in vivo by inoculating both tuber types with the storage pathogens Fusarium sulphureum, F . solani var. coeruleum, Phoma exigua var. foveata and Polyscytalum pustulans . Significantly reduced growth rates and spore and conidiophore numbers were recorded when pathogens were cultured on freeze-dried powder derived from aerial tubers. No effects on spore size of F . solani var. coeruleum were recorded but spore and conidiophore lengths of remaining pathogens were reduced and width increased, except for Colletotrichum coccodes , where the diameter of acervuli increased, and P . exigua var. foveata , where spore length and width were reduced. Reduced disease incidence, severity and spore numbers were recorded on aerial compared with subterranean tubers inoculated in vivo with the four storage pathogens. Spore length of P . pustulans was reduced and spore width increased, while spore length and width of P . exigua var. foveata were reduced on aerial compared with subterranean tubers inoculated with these pathogens. No effects on spore size of Fusarium spp. were recorded. Concentrations of glycoalkaloids, chlorogenic acid, calcium and magnesium were higher in aerial than in subterranean tuber tissue, which may have contributed to increased resistance and effects on size and morphology of fungal spores. The implications of using aerial tubers as an alternative technique for the production of seed potatoes and their importance to the potato industry are discussed.     

Development of resistance to thiabendazole in Helminthosporium solani (silver scurf) as a result of potato seed tuber treatment

Applying thiabendazole to potato seed tubers affected with silver scurf caused by Helminthosporium solani sensitive to thiabendazole decreased the severity of disease on progeny tubers at harvest, but about 50% of the isolates from these were resistant to the fungicide. The disease was not decreased when samples of the progeny tubers were treated with thiabendazole and planted in the following year, and the incidence of resistant isolates increased. Resistant isolates continued to be present when tubers were planted in the next 2 years without fungicide treatment. Treatment with a mixture of thiabendazole and imazalil also decreased the disease and fewer isolates were resistant than when treated with thiabendazole alone, although the proportion increased after treatment with the mixture in the following year. When seed tubers were infected with thiabendazole-resistant H. solani , silver scurf on progeny tubers was not affected by thiabendazole applied to the seed tubers but was decreased by the mixture of thiabendazole and imazalil. Imazalil was equally effective against H. solani sensitive or resistant to thiabendazole.
Some isolates of H. solani had grey aerial mycelium and of 516 of these isolates obtained in 4 years 29% were resistant to the fungicide. Other isolates produced small, black colonies and their frequency increased with thiabendazole treatment of seed tubers. Of 244 of these isolates, 62% were resistant.     

Resistance to thiabendazole in isolates of Helminthosporium solani, the cause of silver scurf disease of potatoes

Isolates of Helminthosporium solani were obtained from potato tubers and cultured on agar containing thiabendazole. Some grew on agar containing 100 mg/l and these were classified as resistant, whereas sensitive isolates produced no mycelial growth on agar containing 5 mg/l. Isolates were also found that were intermediate in their sensitivity to the fungicide. All isolates from Scottish virus tested stem cutting stocks (VTSC) on two farms were resistant whereas those from another farm were sensitive to the fungicide. The proportion of resistant isolates from eight English once-grown seed stocks ranged from 0 to 96%. There has been no previous report of fungicide resistance in H. solani and the possible reasons for these results and their implications are discussed.     

Gas chromatography–mass spectrometry analyses of volatile organic compounds from potato tubers inoculated with Phytophthora infestans or Fusarium coeruleum

Volatile organic compounds (VOCs) collected from potato tubers inoculated with Phytophthora infestans (late blight), Fusarium coeruleum (dry rot) or sterilized distilled water (as a control) were analysed using gas chromatography–mass spectrometry (GC–MS) and gas chromatography–flame ionization detection (GC–FID). A total of 52 volatiles were identified by GC–MS in the headspaces above P. infestans- and F. coeruleum- inoculated tubers after incubation for 42 days in the dark at 10°C. Of these VOCs, the six most abundant were common to both pathogens. These were benzothiazole (highest abundance), 2-ethyl-1-hexanol (second highest abundance), and at approximately equal third abundance, hexanal, 2-methylpropanoic acid-2,2-dimethyl-1-(2-hydroxy-1-methylethyl)-propyl ester, 2-methylpropanoic acid-3-hydroxy-2,4,4-trimethyl-pentyl ester and phenol. In addition, styrene also occurred at approximately equal third abundance in the headspace of F. coeruleum- inoculated tubers, but at lower abundance in the headspace of P. infestans- inoculated tubers. Some VOCs were specific to each pathogen. Butanal, 3-methylbutanal, undecane and verbenone were found at low levels only in the headspace of tubers inoculated with P. infestans , while 2-pentylfuran and copaene were found only in the headspace of tubers inoculated with F. coeruleum . Additionally GC–FID analysis identified ethanol and 2-propanol in the liquid exudate from both P. infestans - and F. coeruleum -inoculated tubers after incubation for 35 days, and in the headspace after incubation for 42 days. These data provide key information for developing a sensor-based early warning system for the detection of postharvest diseases in stored potato tubers.     

Distribution of polygalacturonase, total phenolic substances, polyphenol oxidase and peroxidase in rot zones in sweet potato

Botryodiplodia theobromae causes a spreading rot of sweet potato, while Botrytis cinerea, Phoma exigua var. foveata and Fusarium solani var. coeruleum cause rots of limited extent. Cladosporium cucumerinum does not rot sweet potato tuber tissue. Whichever of these fungi is inoculated into tubers, a zone at the margin of the lesion can be clearly distinguished, from slightly affected but uninfected tissue on one side and infected rotted tissue on the other, by its high content of phenolic substances and polyphenol oxidase and peroxidase activities. Polygalacturonase is present in this zone for spreading B. theobromae lesions but not for the limited lesions caused by the other fungi. Total content of phenolic substances is generally greater in and around limited lesions.     

Survey of benomyl resistance in Fusavium species on winter wheat in England and Wales in 1986

In 1986, samples from 109 winter wheat crops in England and Wales were examined for benomyl resistance in Fusarium species. Of the 704 isolates obtained, 581 were identified as Fusarium nivale, of which 92.1 % were resistant to benomyl. Of the remaining isolates, 82 were identified as F. avenaceum, 40 as F. culmorum and one as F. poae; none of these isolates were resistant to benomyl. The vast majority of isolates of Fusarium spp. were obtained from superficial nodal or internodal lesions on the stems.     

Isozyme and RAPD-PCR analyses of Fusarium avenaceum strains from Finland

Differences in isozyme and RAPD-PCR polymorphisms amongst 33 isolates of Fusarium avenaceum were compared using native polyacrylamide gel electrophoresis and agarose gel electrophoresis. The isolates were collected from different regions of Finland. Amongst eight enzymes analysed clear isozyme polymorphism was detected in five enzymes which could be grouped into 20 different electrophoretic phenotypes and three main groups at the similarity level of 70% in unweighted pair group method with arithmetic average (UPGMA) analysis. RAPD-PCR analysis differentiated all F. avenaceum strains from each other. The phenotypes resulting from RAPD-PCR analysis were grouped into five main groups by UPGMA analysis at the similarity level of 55%. These main groups had several similarities with the main groups from isozyme analysis. RAPD-PCR patterns of 16 isolates of Fusarium graminearum F. culmorum F. equiseti F. oxysporum and F. redolens were also studied and strains from each Fusarium species formed individual groups in UPGMA and principal components analyses. Thus, the extent of isozyme and RAPD-PCR polymorphisms found in Fusarium strains potentially provides a method for identifying the fungi both at strain and species level.     

Fusarium graminearum as a dry rot pathogen of potato in the USA: prevalence,comparison of host isolate aggressiveness and factors affecting aetiology

A 2004–2005 survey of potatoes from stores in the north‐central potato‐producing region of the USA showed that the predominant causes of dry rot were Fusarium graminearum and Fusarium sambucinum. Isolates of F. graminearum originally isolated from potato tubers with dry rot (n = 15), wheat kernels with scab (n = 15), and sugarbeet tap roots with decay (n = 5) were tested for aggressiveness to potato tubers. There were no significant differences in aggressiveness among isolates of F. graminearum, regardless of original host, as measured by their ability to cause dry rot. These findings may have implications for survival of F. graminearum inoculum since potatoes, wheat and sugarbeets are frequently used in crop rotation in the region. Fusarium graminearum required larger wounds for infection of potato tubers than F. sambucinum. Plug‐removal injury, simulating a stolon‐removal injury, resulted in equal incidence of dry rot caused by the two Fusarium species, whereas abrasion and bruising injury were sufficient for infection and dry rot development by F. sambucinum, but not F. graminearum. A change in harvest practices from vine‐killing prior to harvest to mechanical vine‐killing on the day of harvest may be a factor affecting the onset of dry rot caused by F. graminearum, since this process often causes large wounds at the stem end of the tubers when the stolon is forcibly removed.     

Phytophthora infestans Populations from Tomato and Potato in North Carolina Differ in Genetic Diversity and Structure

ABSTRACT Phytophthora infestans causes a destructive disease on tomato and potato. In North Carolina (NC) potatoes are mostly grown in the east, whereas tomatoes are grown in the mountainous areas in the western part of the state. Five genotypes of P. infestans were identified from 93 and 157 isolates collected from tomato and potato over a 5 year period between 1993 and 1998. All isolates collected from potato in eastern NC were the US-8 genotype, whereas only a single isolate was the US-1 genotype. Tuber blight was found on immature daughter tubers in a single field in 1997, however infection on mature tubers was not observed. Within potato fields, a range of sensitivity to metalaxyl was observed among isolates but all were either intermediate or highly resistant to the fungicide. In contrast, isolates from tomatoes included previously reported US-7 and US-8 genotypes and two new genotypes called US-18 and US-19 (A2 mating type, allozyme genotype Gpi 100/100 and Pep 92/100). These genotypes had unique restriction fragment length polymorphism banding patterns, were sensitive to metalaxyl, and have not been reported elsewhere. All genotypes, with the exception of the US-1, were the Ia mitochondrial haplotype. Thus, isolates of P. infestans from tomato were more genetically diverse over time in NC than those from potato and include two new genotypes that are sensitive to metalaxyl.     

Bacterial and fungal diseases in the major potato-growing areas of Tunisia

In a two year survey of Tunisian rustic potato stores, the losses were nearly equally distributed between dry rots induced by Fusarium solani and watery wound rot or leak caused by Pythium aphanidermatum and P. ultimum. Symptoms of leak were different from those normally induced by Pythium spp. Surveys of the growing potato crop confirmed the prevalence of these pathogens, which caused wilt and internal vascular stem necrosis. Most rotted progeny tubers also exhibited leak symptoms caused by P. aphanidermatum. Hardly any Erwinia diseases were found in Tunisia. The incidence of diseased plants was significantly higher in fields with successive cultivation of solanaceous crops, which may be attributed to a high rate of soil infestation by wilt-inducing pathogens such as Pythium spp., F. solani and Verticillium dahliae. The health of seed potatoes also played a significant role, affecting the incidence of rot in potato stores as well as in the soil before plant emergence. Therefore, Tunisian integrated management programmes for potato diseases should focus on soil disinfestation with appropriate crop rotation as well as seed quality and treatments.     

Metalaxyl sensitivity of isolates of Phytophthora erythroseptica in Prince Edward Island

Isolates of Phytophthora erythroseptica , causal agent of pink rot of potato, were obtained from diseased tubers collected in Prince Edward Island (PEI), Canada. In an in vitro assay of field and single-zoospore isolates, all isolates of the pathogen from PEI were sensitive to metalaxyl (EC₅₀ < 0·5  µ g mL⁻¹), unlike moderately or highly resistant reference isolates obtained from Maine, USA. Allozyme-banding patterns at the glucose-6-phosphate isomerase ( Gpi ) locus were identical (91/91) for all isolates of P. erythroseptica examined from PEI and Maine, but could be used to distinguish isolates of P. erythroseptica from local isolates of Phytophthora infestans . Inoculation of potato tubers from plants treated with foliar applications of metalaxyl in the field indicated that compounds inhibitory to metalaxyl-sensitive isolates of P. erythroseptica were present in the periderm, even after 4 months' storage. By contrast, moderately or highly resistant isolates of the pathogen caused significantly ( P  ≤ 0·05) more disease than sensitive isolates in tubers from plants treated with metalaxyl. The effectiveness of metalaxyl in controlling pink rot in a particular region will depend on resistance levels of local populations of P. erythroseptica . Preventing the development and spread of resistant strains of P. erythroseptica will be critical in maintaining metalaxyl as an effective chemical for control of this pathogen.     

Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.     

禾谷镰刀菌对苯基吡咯类杀菌剂咯菌腈的抗性机制

由禾谷镰刀菌引起的小麦赤霉病是世界小麦生产上的重要真菌病害。为了进一步明确禾谷镰刀菌对苯基吡咯类杀菌剂咯菌腈产生抗性的机制,本文以前期室内通过药剂驯化方式得到的4株禾谷镰刀菌对咯菌腈的高水平抗性突变体 (其抗性倍数在318.2~782.9之间) 为主要研究材料,采用生物测定及分子生物学等方法开展了禾谷镰刀菌对咯菌腈的抗性机制研究。结果表明:供试禾谷镰刀菌抗咯菌腈突变体对小麦幼穗的致病力降低了约50%,部分菌株 (2XZ-4R) 甚至完全丧失了对小麦的致病能力;抗性突变体对渗透胁迫 (0.5 mol/L NaCl, 1.0 mol/L MgCl₂, 1.0 mol/L葡萄糖或1.0 mol/L甘露醇) 高度敏感,且菌丝生长抑制率较敏感菌株降低约50%以上,表明其环境适合度显著下降。同时,抗性突变体中苯丙氨酸解氨酶 (PAL)、过氧化物酶 (POD) 和多酚氧化酶 (PPO) 活性较敏感菌株均升高2倍以上。分子生物学分析发现,供试抗性突变体中候选靶标基因 (FgOs1和FgOs5) 的表达量显著下调 (P<0.05),推测FgOs1和FgOs5可能参与了禾谷镰刀菌对咯菌腈抗性的形成过程。总之,该研究探究了禾谷镰刀菌抗咯菌腈突变体的生物学特性,并为深入揭示禾谷镰刀菌对咯菌腈的抗性分子机制提供了新的思路。     

甘肃省马铃薯镰刀菌干腐病优势病原的分离鉴定

 为明确甘肃马铃薯镰刀菌干腐病的优势病原,2006 年12 月~ 2007 年3 月由西至东从甘肃张掖、天祝、永登、临洮、渭源和西和等6 县市的马铃薯贮藏窖中采集表现有镰刀菌干腐病症状的马铃薯薯块,以组织分离法分离病原,单孢纯化镰刀菌(Fusarium spp. )菌株后,以形态学为基础,参照Nelson 镰刀菌分类系统进行鉴定。结果表明:6 个采样区共分离到293株镰刀菌菌株,其中以接骨木镰刀菌(F. sambucinum)和茄病镰刀菌(F. solani)出现频率高,是优势种。分析发现第一优势种随采样区而异,张掖、天祝和渭源采集的样品中茄病镰刀菌分离频率分别为42. 6% 、42. 1% 和32. 4% ,接骨木镰刀菌分离频率分别为14. 8% 、5. 3% 和26. 5% ,茄病镰刀菌为第一优势种;永登、临洮和西和采集的样品中接骨木镰刀菌分离频率分别为52郾1% 、50. 9% 和55. 2% ,茄病镰刀菌的分离频率分别为23. 3% 、32. 7% 和20. 7% ,接骨木镰刀菌为第一优势种。本文进一步对其在PDA、CLA 上的培养特征进行了观察和描述。按照柯赫氏法则用混合菌株接种法对大西洋(Atlantic)、夏波蒂(Shepody)以及一地方品种进行致病性测定,证实了优势菌种对块茎的致病性。利用EF鄄1琢基因引物(EF鄄1H 和EF鄄2T)对接骨木镰刀菌菌株GAUF鄄F12 进行基因组DNA 的PCR 扩增,将PCR 产物回收测序后在GenBank 上比对,菌株GAUF鄄F12 与GenBank 登记的接骨木镰刀菌5 个菌株的同源性均达99% ;用DNASTAR 分析软件将同源性较高的登记菌株的序列与GAUF鄄F12 菌株构建同源性树,结果表明:该菌株与以上5 个接骨木镰刀菌菌株均位于同源性树的同一分支,聚为一类,与形态学的鉴定结果一致。