Lack of biocontrol capacity in a non-pathogenic mutant of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis>

The aim of this study was to assess the biocontrol capacity of rev157, a non-pathogenic mutant of a pathogenic strain of Fusarium oxysporum f. sp. melonis (Fom24). Inoculated in association with the virulent parental strain, the mutant rev157 did not protect the host plant (muskmelon) against infection by Fom24. Applied on flax, a non-host plant, the mutant rev157 was not able to protect it against its specific pathogen F. oxysporum f. sp. lini. On the contrary the parental strain Fom24 did protect flax as well as a soil-borne biocontrol strain (Fo47). Since the mutant rev157 was affected neither in its growth in vitro nor in its capacity to penetrate into the roots, it can be speculated that the mutation has affected traits responsible for interactions within the plant. In F. oxysporum the pair of strains Fom24/rev157 is a good candidate to identify genes involved in the biocontrol capacity of F. oxysporum and to test the hypothesis of a link between capacity to induce the disease and capacity to induce resistance in the plant.     

Remodelling of actin cytoskeleton in tomato cells in response to inoculation with a biocontrol strain of Fusarium oxysporum in comparison to a pathogenic strain

Knowing that actin microfilaments play a key role in the mobilization of the defence‐associated cellular responses, the aim of this study was to compare changes affecting the actin cytoskeleton in tomato cells after inoculation with germinated microconidia of a biocontrol (Fo47, Fom24) or a pathogenic (Fol8) strain of Fusarium oxysporum. Actin microfilaments were observed by labelling with TRITC‐phalloidin combined with fluorescence microscopy. Results showed that only tenuous changes in the actin cytoskeleton architecture occurred after inoculation with the biocontrol strains whereas the actin cytoskeleton was significantly altered after inoculation with the pathogenic one. In the two types of interaction, cell death occurs and can be considered as one key component of cell defence responses. A pharmacological approach using cytochalasins was chosen to determine whether the inhibition of actin polymerization differently affects the kinetics of tomato cell death. Data showed that cytochalasins reduced cell death induced by the biocontrol strain Fo47, and in contrast, increased cell death induced by the pathogenic strain Fol8, suggesting that the pathway leading to cell death differs in the protective and compatible interactions.     

Bacillus subtilis as an alternative biologically based strategy for controlling Fusarium wilt disease in tomato: A histological study

The employment of formulateBacillus subtilis as a biocontrol agent successfully controlledFusarium oxysporum f.sp.lycopersici within tomato seedlings (in vivo). B. subtilis was able to proted cortex and vascular tissues of tomato against progression of the wilt pathogen. No changes were observed in tomato tissues due to application ofB. subtilis except for hypertrophy and elongation of cortex tissues, which indicates the production of plant growth hormones byB. subtilis.     

Lipid metabolism in tomato and bean as a sensitive monitor for biocontrol of wilt diseases

The lipids metabolism of tomato and bean plants during biological control of wilt pathogens (Fusarium oxysporum f.sp.lycopersici andF. oxysporum f.sp.phaseoli, respectively) byBacillus subtilis was investigated. The interaction of wilt pathogens with both tomato and bean caused an imbalance and drastic reduction in total lipids, triacylglycerol, sterol and all phospholipd fractions except phosphatidic acid. The application of a formulated biocontrol agent,B. subtilis, eliminated the detrimental effect of both wilt pathogens and consequently prevented catabolism of lipid fractions in both tomato and bean. Moreover, the changes in the lipid fractions as a sensitive monitor for biocontrol of wilt diseases suggest a positive correlation between the application ofB. subtilis and improvement in the host metabolism towards anabolism. http://www.phytoparasitica.org posting Sept. 20, 2006.     

复合微生物菌剂对苹果再植病害的生防效果测定及拮抗菌株鉴定

为探究复合微生物菌剂对苹果再植病害的生防效果,以苹果砧木实生海棠幼苗为试材,通过盆栽试验测试了木美土里复合微生物菌剂对苹果再植病害的防治效果及对再植海棠的促生作用,然后对该菌剂的可分离微生物进行了分离纯化,并以尖孢镰刀菌Fusarium oxysporum菌株HS2为靶标菌,测试分离获得的10株菌株的拮抗效果,并对拮抗效果显著的1株真菌菌株和2株细菌菌株进行种类鉴定。结果表明,木美土里复合微生物菌剂对苹果再植病害的防治效果达87.78%;经该菌剂处理后,再植海棠苗的株高、茎粗、鲜重、干重及叶面积分别是对照处理的1.51、2.00、5.74、5.21和2.83倍,表明该菌剂有显著的促生作用。木美土里复合微生物菌剂对菌株HS2的抑制率达到68.59%,并从中分离到7株真菌菌株,3株细菌菌株。其中,拮抗效果高于80.00%的真菌菌株有1株,细菌菌株有2株。经鉴定,真菌菌株MMTL-1为哈茨木霉Trichoderma harzianum;细菌菌株BM-01和BM-03均为解淀粉芽胞杆菌Bacillus amyloliquefaciens。     

基于链特异性RNA-seq的禾谷镰刀菌全生活史转录组分析

为明确植物病原真菌禾谷镰刀菌Fusarium graminearum全生活史的转录组特征和基因表达模式,采用生物信息学技术对其生活史不同阶段15个时期或组织的链特异性RNA-seq数据进行分析。结果表明,共有8 106个基因在所有时期均有表达,为禾谷镰刀菌生活史核心基因,占总基因数的47.2%;有性生殖和侵染过程中表达的基因数相对较多,其中有性生殖后期表达的基因数最多,达15 221个;无性产孢和侵染小麦穗过程中基因表达水平整体较高,而分生孢子中基因表达水平最低。在燕麦培养基上禾谷镰刀菌气生菌丝的基因表达模式与侵染过程中的基因表达模式较为相似,而与营养生长菌丝的基因表达模式差异较大。该菌次生代谢物合成特征酶基因和分泌蛋白基因的表达模式均分成3类,即分别在侵染、营养生长和有性生殖过程上调表达,暗示其在生活史不同阶段的特异性功能。     

Extracts of killedPenicillium chrysogenum Induce resistance against Fusarium wilt of melon

Dry fungal biomass ofPenicillium chrysogenum (dry mycelium), a waste product of the pharmaceutical industry, was extracted with water and applied to the roots of melon plants before or after inoculation withFusarium oxysporum f.sp.melonis (Font). Seedlings (4–6 days after emergence) treated with either acidic dry mycelium extract (DME) or neutralized dry mycelium extract (NDME) were protected against challenge infection withFom. A single drench with 2–5% DME applied 12–72 h before inoculation provided significant control of the disease compared with water-drenched, challenged seedlings. No protection was seen in plants treated 0–6 h before inoculation or 0–48 h after inoculation. Neither DME nor NDME (0.5–5%) had any effect on fungal growthin vitro, which implied that disease controlin vivo was mediated by induced resistance. The resistance induced by DME protected melon plants not only against race 1,2, but also against the three other races of the pathogen, indicating a race-non-specific resistance againstFom. Both DME and NDME significantly increased peroxidase activity and free L-proline content in seedlings 12 h and 48 h after soil drench, respectively. Resistance to Fusarium wilt was significantly associated with elevated levels of peroxidase activity but not with free L-proline content. Thus, peroxidase might be involved in the defense mechanisms activated by DME or NDME. http://www.phytoparasitica.org posting Aug. 31, 2001.     

紫花苜蓿根际芽胞杆菌YB-2的鉴定及其生防和促生能力分析

为获得对紫花苜蓿Medicago sativa生长有益的生防菌,以尖孢镰刀菌Fusarium oxysporum为指示菌从紫花苜蓿根际土壤中分离筛选拮抗菌株,基于形态学特征、生理生化特性和16S rDNA与gyrB基因序列分析对其进行鉴定,并评价其对紫花苜蓿根腐病的生防效果以及提高寄主植物耐盐碱胁迫的能力。结果表明,从紫花苜蓿根际土壤中筛选到1株对尖孢镰刀菌有较强拮抗作用的菌株YB-2,该菌株对尖孢镰刀菌菌丝生长的抑制率为71.37%;结合形态学特征、生理生化特性和16S rDNA与gyrB基因序列分析结果将菌株YB-2鉴定为枯草芽胞杆菌Bacillus subtilis。菌株YB-2具有溶解无机磷的能力,且具有产氰化氢、羧甲基纤维素酶、1-氨基环丙烷-1-羧酸脱氨酶、吲哚乙酸和嗜铁素的能力,在NaCl含量为1%～9%、pH 7～9条件下均能生长。菌株YB-2对紫花苜蓿根腐病的相对防效达56.83%;在盐碱条件下,与对照相比,接种YB-2的紫花苜蓿株高极显著增加了17.82%,根长显著增加了28.22%,地上部鲜重和地上部干重分别显著增加了51.12%和48.57%。表明菌株YB-2兼...     

Identification of differentially expressed genes in the mutualistic association of tall fescue with Neotyphodium coenophialum

Tall fescue (Lolium arundinaceum), an agronomically important forage grass, is typically associated with a mutualistic asexual fungus Neotyphodium coenophialum. Plant colonization is endophytic with no symptoms, and fungal growth is confined to the intercellular spaces. The endophyte enhances host fitness by providing protection from various abiotic and biotic stresses and by improving nutrient acquisition. By suppression subtractive hybridization (SSH) we identified 29 genes that are up-regulated or down-regulated in endophyte-infected tall fescue as compared to endophyte-free tall fescue. Of the genes that had matches to known genes present in the NCBI databases (approximately 50%), several had roles related to plant defense and stress tolerance. Differential expression of these genes was confirmed by semi-quantitative RT-PCR, competitive RT-PCR, and northern hybridization. Endophyte-associated changes in gene expression patterns were consistent among cultivars of tall fescue but differed in some other grass–endophyte associations. Our results indicate that both partners in this symbiosis are active participants, and that the endophyte may be suppressing at least one plant defense gene (putatively encoding PR-10). Further analyses of the differentially expressed genes should aid in understanding the fundamental nature of this mutualistic symbiosis and provide insight into the mechanisms of documented endophyte-enhanced plant improvements.     

Three evolutionary lineages of tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici, based on sequences of IGS, MAT1, and pg1, are each composed of isolates of a single mating type and a single or closely related vegetative compatibility group

Three evolutionary lineages of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici were found among a worldwide sample of isolates based on phylogenetic analysis of the ribosomal DNA intergenic spacer region. Each lineage consisted of isolates mainly belonging to a single or closely related vegetative compatibility group (VCG) and a single mating type (MAT). The first lineage (A1) was composed of isolates VCG 0031 and MAT1-1; the second (A2) included VCG 0030 and/or 0032 and MAT1-1; and the third (A3) included VCG 0033 and MAT1-2. Race 1 and race 2 isolates belonged to the A1 or A2 lineages, and race 3 belonged to A2 or A3 lineages, suggesting that there is no correlation between race and lineage. However, for the isolates from Japan, race 1 (with one exception), race 2, and race 3 isolates belonged to A2, A1, and A3 lineages, respectively. These results suggest that the races could have evolved independently in each lineage; and in Japan the present races were likely to have been introduced independently after they had evolved in other locations.     

一株生防内生真菌的分离筛选、鉴定及抑菌特性

为获取优良植物病害生防菌,以交链孢菌Alternaria tenuis Ness和尖镰孢菌Fusarium oxysporum Schlecht为指示菌,采用平板对峙培养法、抑菌圈法和菌落直径法对健康龙柏鳞叶小枝中分离的4株优势内生真菌进行了筛选评价,并结合形态学观察及ITS序列分析对筛选出的生防内生真菌进行了鉴定。结果表明,对峙培养发现1菌株对交链孢菌和尖镰孢菌均具有较好的抑制作用,抑菌率分别为52.05%和48.65%,标记其为LB-1;LB-1菌株与指示菌对峙培养的菌落交界面处,2种指示菌的菌丝均出现了消解、断裂,而LB-1的菌丝则生长正常。未灭菌LB-1菌株发酵液对交链孢菌和尖镰孢菌产生的抑菌圈直径分别为14.1 mm和13.7 mm,抑菌率分别为47.89%和48.72%;灭菌LB-1菌株发酵液对交链孢菌和尖镰孢菌的抑菌圈直径分别为15.3 mm和15.0 mm,抑菌率分别为5.96%和33.90%,指示菌在生长过程中菌丝溶解现象明显。经鉴定,LB-1菌株为毛壳菌属Chaetomium中的近缘毛壳菌C.subaffine,是一种极具生防潜力的内生真菌,其抑菌特性可能通过溶菌作用实现。     

Pisatin demethylation by fungal pathogens and nonpathogens of pea: association with pisatin tolerance and virulence

Previous studies have indicated that detoxification of their hosts' phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and virulence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates ofPythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts' phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized enzyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cytochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating population VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share limited nucleotide similarity with N. haematococca PDA genes.     

一株对桃蚜有高致病性球孢白僵菌的分离、筛选与鉴定

为筛选出对桃蚜Myzus persicae具有高致病性的生防真菌,以从陕西省秦岭原始森林采集到的鳞翅目僵虫虫体中分离获得的5株真菌为研究对象,测定其对桃蚜的致病性并筛选出高致病性菌株表现最佳杀蚜活性时的孢子悬浮液浓度,同时结合形态学及18S rDNA和ITS-rDNA序列分析对致病性最高的病原菌进行鉴定。结果显示,从5株菌株中初步筛选出1株高致病性菌株BQ-63,处理7 d后桃蚜的死亡率为80.33%,校正死亡率为81.58%,僵虫率也达到最高,为80.78%;当菌株BQ-63的孢子浓度为108个/mL时,对桃蚜的致病性达到最高,处理7 d后死亡率为89.53%,校正死亡率为90.10%,僵虫率为89.84%;通过形态学和分子生物学鉴定,确定菌株BQ-63为球孢白僵菌Beauveria bassiana。表明菌株BQ-63对桃蚜具有高致病性,可作为生防真菌进行进一步的研究。     

Pathogenicity and RAPD analysis of Phytophthora nicotianae pathogenic to pepper in Tunisia

Nine isolates of Phtophthora nicotianae were isolated from infected pepper plants. Their pathogenicity was studied in Capsicum annuum in comparison with P. nicotianae isolates from tomato and tobacco. The pathogenicity test showed that pepper isolates of P. nicotianae are adapted to their host. Banding patterns obtained by RAPD analysis with six oligonucleotide primers revealed polymorphism that grouped the isolates independently of the plant host. The polygenic dendrogram showed that pepper isolates were more similar to tomato isolates than to tobacco isolates. The RAPD bands of 1300 and 1500 bp, detected with primers OPD-01 and OPD-10, respectively, appeared specific to the most pathogenic pepper isolates. The OPK-08-1950 seems specific to the isolates of P. nicotianae from tomato. These results suggest that host specified might occur in P. nicotianae and that may be due to interspecific hybridization events resulting in novel pathogenic behavior.     

球孢白僵菌定殖提高番茄对灰霉病抗性及其作用机理

为明确球孢白僵菌Beauveria bassiana定殖对番茄植株抗病性的影响及作用机理,采用灌根法将球孢白僵菌芽生孢子悬浮液接种于番茄植株内,并通过人工接种灰霉菌Botrytis cinerea评价球孢白僵菌定殖后番茄对灰霉病的抗性水平;检测灰霉菌胁迫下番茄植株不同位置叶片内球孢白僵菌的相对含量;测定番茄叶片内草酸氧化酶（oxalate oxidase,OXO）、几丁质酶（chitinase,CHI）和ATP合成酶（ATP synthase,atpA）3种抗病相关基因的表达量。结果表明,球孢白僵菌定殖能够提高番茄植株对灰霉病的抗性,接种灰霉菌第5天,番茄植株发病率、病斑直径和病情指数分别下降了61.6%、41.4%和26.4%;在灰霉菌胁迫下番茄植株内球孢白僵菌偏好于在病原菌感染位置定向聚集,并且引起植物抗病基因OXO、CHI和atpA的表达量上调。表明球孢白僵菌能通过内生定殖与植物互作提高植物抗病性,在植物病害生物防治领域有较大应用潜力。     

棉隆与淡紫拟青霉联合防治番茄根结线虫病的效果评价

为明确土壤熏蒸与生防微生物对番茄根结线虫病的联合作用效果,采用移栽前棉隆熏蒸处理后穴施淡紫拟青霉Paecilomyces lilacinus YES-2-14生防菌剂的方法在温室大棚开展了联合防治试验。结果表明,棉隆用量为35 g/m~2时,24周拉秧时线虫数量减少90%以上,防效达到85.5%;移栽时穴施浓度10~7~10~8CFU/g的淡紫拟青霉菌剂,24周拉秧时防效为44.4%~59.6%;而当线虫初始密度达到674头/100 g土时,单一菌剂防效只有12.5%,但番茄长势和产量显著提高。棉隆熏蒸后再联合施用淡紫拟青霉菌剂,2个月和5个月时防效比单一棉隆处理分别提高14.1%和21.8%,比单一菌剂处理提高14.3倍和4.1倍,产量较对照、棉隆熏蒸和单一菌剂处理分别提高22.1%、9.5%和2.4%,且该处理地块线虫数量始终最低,与对照差异显著,而淡紫拟青霉数量则高于单一淡紫拟青霉菌剂处理。表明棉隆和淡紫拟青霉菌剂联合使用不仅能显著降低线虫数量,有效防治番茄根结线虫病,还能够促进植株生长并提高产量。     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

Ultrastructural and cell wall modifications during infection of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis> roots by a pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain

Fusarium species are soil-borne fungal pathogens that produce a variety of disease symptoms when attacking crop plants. The mode of root colonization of Eucalyptus viminalis seedlings by a pathogenic F. oxyporum strain (Foeu1) at the ultrastructural level and changes in cell wall pectin during host pathogen interactions are described. Root systems of E. viminalis plants were inoculated with F. oxysporum in an in vitro model system. Hyphae of F. oxysporum adhered to the outer epidermal cell walls through fibrillar material, and after penetration they spread into the internal tissues. They developed intercellularly and intracellularly in the root cortex and invaded vascular tissues. Papillae were induced, and the host plasma membrane ruptured in colonized cells, causing rapid host tissue and cell damage. Changes in distribution and occurrence of nonesterified and methyl-esterified pectins were evaluated after root colonization by F. oxysporum using two monoclonal antibodies, JIM 5 and JIM 7, respectively. Nonesterified pectin in control roots was mainly localized in the epidermal cell walls and middle lamellae in parenchymal cortex, whereas methyl-esterified pectin accumulated more in primary cell walls of the cortex and phloem. Decreases in immunodetected nonesterified and methyl-esterified pectins were associated with extensive plant tissue degradation after root colonization by the pathogenic fungus.