Effect of Relative Humidity on Sporulation of Fusarium oxysporum in Various Formulations and Effect of Water on Spore Movement Through Soil

ABSTRACT Fusarium oxysporum f. sp. erythroxyli is being investigated as a mycoherbicide for the narcotic plant coca. Sporulation of the fungus in seven formulations containing different organic substrates and movement of its propagules through soil were studied. The formulations were a granular wheat flour/kaolin (pesta); an extruded wheat and rice flour (C-6); and five alginate pellet products containing corn cobs, soybean hull fiber, canola meal, rice flour, or rice flour plus canola oil. Formulations were incubated at 25 degrees C for 6 weeks in desiccators with various salt solutions to provide nine relative humidities (RH), ranging from 100% (pure deionized water) to 0% (anhydrous (CaSO(4)). Hyphae of F. oxysporum f. sp. erythroxyli grew out of alginate pellets with canola meal, rice, and rice plus canola oil as early as 24 h at 100% constant RH. Alginate pellets of rice plus canola oil and granular C-6 and pesta formulations consistently produced more microconidia, macroconidia, and CFU than the other four formulations at all RH tested. The C-6 formulation produced more propagules than the other formulations at low RH (<53%). Canola meal pellets produced more spores than three other formulations when exposed to fluctuating RH (100 to 75%). The effect of percolating water on spore movement through soil was studied using a plant-pathogenic isolate of F. oxysporum f. sp. niveum. To determine the effect of water percolation on propagule movement, formulations were placed on soil columns and artificial rain was applied. In general, 10-fold fewer CFU were recovered at a 8- to 10-cm depth compared with a 0- to 2-cm depth.     

Effects of the fungus Crinipellis perniciosa, causal agent of witches' broom disease, on cell and tissue cultures of cocoa (Theobroma cacao L.)

The symptoms of witches' broom disease in cocoa, caused by the Basidiomycete fungus Crinipellis perniciosa , are pronounced swelling of the terminal and axillary buds followed in the long term by necrosis of this tissue. The direct effect of C. perniciosa on cocoa cells was examined under controlled conditions by growing primary and secondary phase cultures of the fungus separately and also with callus cultures and with cell suspensions. Both primary and secondary phase mycelium reduced growth of callus cultures by about 47% after one week compared with the controls. However, cell suspensions containing primary phase mycelium showed initial growth double that of the uninfected controls after 5 days, but then growth was reduced below that of the control and particularly when the primary phase became secondary phase mycelium. This change in fungal development coincided with the time that the cell culture reached the stationary growth stage. Cell cultures inoculated with stationary phase mycelium showed the same growth as the control after 5 days but then growth was reduced to 50% of the control after 19 days incubation and remained at this low level subsequently. The inhibitory effect of secondary phase mycelium was examined by incubating callus and cell suspensions with culture filtrate from liquid cultures of the secondary phase. Inclusion of 50% by volume of culture filtrate from the secondary phase in the growth medium for callus and cell suspensions, respectively, resulted in a reduction in growth of the plant tissue cultures. Addition of fungal culture filtrates also led to loss in potassium and loss of viability of cell suspensions and of isolated cells as represented by protoplasts. The necrotrophic mode of the secondary phase may be achieved through the production of phytotoxins acting on the host cell membrane.     

拮抗性链霉菌对大丽轮枝菌微菌核形成与萌发的影响

为探索拮抗性链霉菌对棉花黄萎病病原大丽轮枝菌Verticillium dahliae Kleb.的抑菌机制,采用菌丝生长速率法、微菌核萌发法研究了6株拮抗链霉菌无菌发酵滤液对大丽轮枝菌生长、微菌核形成与萌发的影响。链霉菌无菌发酵滤液对大丽轮枝菌菌落生长、菌核形成和微菌核萌发有明显抑制作用。其中菌株B49的抑菌效果最好,5倍稀释发酵液培养14天时对菌落生长的抑菌率达69.7%;菌株B49、D184和Act12的5倍稀释发酵液对微菌核形成的抑制率达100%;将经B49、D184和Act12发酵液处理后丧失形成微菌核能力的大丽轮枝菌菌株转接至不含发酵液的PDA培养基,连续传代至第5代,其仍然不能恢复形成微菌核的能力;微菌核在含有菌株D184 5倍稀释发酵液的培养基上培养168 h时,萌发率仅为38.3%。     

Antimicrobial Activity of Culture Filtrate of Bacillus amyloliquefaciens RC-2 Isolated from Mulberry Leaves

ABSTRACT A potential antagonist, Bacillus amyloliquefaciens strain RC-2, against Colletotrichum dematium, mulberry anthracnose fungus, was obtained from healthy mulberry leaves by in vitro and in vivo screening techniques. Application of culture filtrate of RC-2 inhibited disease on mulberry leaves, indicating that suppression was due to antifungal compounds in the filtrate. Development of mulberry anthracnose on mulberry leaves was inhibited only when the culture filtrate was applied before fungal inoculation, and it was not inhibited by application after inoculation. These results suggest that the antifungal compounds in the filtrate exhibit a preventive effect on the disease. Peptone significantly increased production of the antifungal compounds. The culture filtrate of RC-2 also inhibited the growth of several other phytopathogenic fungi and bacteria, such as Rosellinia necatrix, Pyricularia oryzae, Agrobacterium tumefaciens, and Xanthomonas campestris pv. campestris, in vitro. From the culture filtrate of RC-2, seven kinds of antifungal compounds were isolated by high performance liquid chromatography analysis, and one of the compounds was determined as iturin A2, a cyclic peptide, by nuclear magnetic resonance and fast atom bombardment mass analysis.     

Toxic culture filtrates produced byCalonectria ilicicola, causal agent of red crown rot of soybean

Eleven soybean cultivars with different levels of susceptibility to virulent isolate SG915 ofCalonectria ilicicola were examined for reaction to metabolites produced by the isolate. When the culture filtrate from isolate SG915 was applied to trifoliates from 11 cultivars, cvs. ‘Cajun’ and ‘Asgrow 7986’ exhibited reduced wilting severity. However, there was no correlation between sensitivity to culture filtrate and susceptibility to the fungal isolate. Wilting severity on cv. ‘Riverside 699’ was greatest when trifoliates were treated with culture filtrates from isolates SG915 (highly virulent) and C31 (less virulent). The dilution end-point for culture filtrates of virulent isolate SG915 was determined to be 1:8. Nonautoclaved culture filtrates caused complete wilt of soybean trifoliates after 36 h, but autoclaved culture filtrates demonstrated a reduced ability to wilt leaves. Electrolyte leakage from treated leaf tissues increased over time regardless of the concentrations of culture filtrate tested. The greatest electrolyte losses were observed during the initial 30 min incubation of leaf tissues. The highest concentration of culture filtrate (50%, v/v) induced more electrolyte loss than the low concentration (10%, v/v) or control. These results suggest that toxic metabolites ofC. ilicicola may be involved in disease development with leaf symptom expression.     

Development and Pathogenicity of the Fungus Crinipellis perniciosa on Interaction with Cacao Leaves

ABSTRACT We investigated developmental changes in the primary mycelium of Crinipellis perniciosa upon its interaction with immature and mature leaves of Theobroma cacao. On nutritive medium, the primary mycelium grew significantly slower in the presence of host tissue than without host tissue. In the absence of the cacao leaves, incomplete phase transition occurred after 5 days, wherein older hyphae progressed to the dikaryotic state and growing tips remained monokaryotic. Phase transition occurred between 3 and 5 days on mature leaves, 10 and 12 days on meristematic leaves, and required 2 weeks on T. cacao callus tissue. The biotrophic mycelia were able to invade immature and mature cacao leaves without open wounds or stomata. Club-shaped hyphal tips and the formation of adhesive structures were induced by cuticle extracts and suggest host recognition. The initial cuticular disintegration at the site of penetration was followed by blister formation and complete digestion of leaves by the primary mycelium. The data suggest specific interactions between host and pathogen that control the onset of the necrotrophic phase of the fungus. The data further indicate that primary mycelium rather than spores can be used to study C. perniciosa pathogenicity.     

Growth of Coniothyrium minitans,Gliocladium roseum,Trichoderma harzianum and T. viride from alginate pellets and interaction with water availability1

Conidia, chlamydospores and mycelia of Coniothyrium minitans, Gliocladium roseum, Trichoderma harzianum and T. viride were obtained from liquid or solid culture and formulated within alginate pellets. Quantitative assessment of these pellets over a 12-week period showed a decrease in the number of colony-forming units (cfu) from about 10⁷ to 10³-10⁴ g^-1 air-dried pellets. Qualitatively, growth was assessed by direct plating the alginate pellets on rich (potato dextrose agar, PDA) and poor (water agar, WA) substrates. Rate of growth (mm day^-1) from individual pellets was decreased by water potential in the range -0.25 to-2.8 MPa, with growth significantly less on WA than PDA for all species except G. roseum. All species grew from 95 to 100% of direct-plated pellets on PDA over the water-potential range tested. However, on WA, interaction between water stress (-2.8 MPa) and lack of nutrients resulted in a reduction, with less than 30% of pellets of some formulations showing growth. In contrast to the quantitative results, there was little change in the growth rate of test species from pellets over a 12-week period, particularly on PDA. Growth from pellets could not be conclusively demonstrated in unsterile soil. The shelf-life and viability of alginate pellets in soil is assessed.     

Use of GUS Transformants of Fusarium subglutinans for Determining Etiology of Mango Malformation Disease

ABSTRACT Fusarium subglutinans has been associated with mango floral and vegetative malformation, although confusion exists regarding the etiology of the disease. A wild-type isolate of F. subglutinans causing mango malformation disease was transformed with the GUS (beta-glucuronidase) reporter and hygromycin resistance genes. Five stable transformants were isolated containing varying copy numbers at different integration sites. Specific GUS activity was quantified for the transformants, whereas no activity was recorded for the wild-type isolate. The transformants and the wild-type isolate were inoculated into healthy mango floral and vegetative buds. Typical symptoms of misshapen shoots with short internodes, stubby leaves, and bunchy, malformed inflorescences were observed 6 to 8 weeks following inoculation. The presence of GUS-stained mycelium of the pathogen viewed microscopically within infected plant organs provided unequivocal evidence that F. subglutinans is indeed a causal agent of mango malformation disease.     

Differential effects of phytotoxic metabolites from <Emphasis Type="Italic">Alternaria tagetica</Emphasis> on <Emphasis Type="Italic">Tagetes erecta</Emphasis> cell cultures

Alternaria tagetica, a fungus that causes early blight in marigold (Tagetes erecta), produces two groups of phytotoxic metabolites: one hydrophilic and the other lipophilic that show phytotoxic activity when tested by the leaf-spot assay in T. erecta. We evaluated the cellular effects of the phytotoxic culture filtrate of A. tagetica and the hydrophilic and lipophilic fractions, then determined whether the filtrate or the fractions differentially induced pathogenesis-related mechanisms in the plant. The culture filtrate and the phytotoxic fractions had adverse effects on cell viability, fresh mass, and the number of cells, and induced ROS accumulation, lipid peroxidation and DNA damage on T. erecta cell suspension cultures, and these effects are related to pathogenic mechanisms attributed to phytotoxins. However, although exposure of marigold cells to the phytotoxic culture filtrate of A. tagetica triggered programmed cell death, the hydrophilic and the lipophilic phytotoxic fractions induced death that was more related to a toxic effect leading to necrosis. This study presents a complementary perspective in the search for the roles of metabolites, including phytotoxins, produced by phytopathogenic fungi during plant infection.     

Antifungal activity of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. culture filtrates against the rice pathogen <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

An ethyl acetate extract of a culture filtrate (ECF) from an unidentified fungal isolate O821 was evaluated for antifungal activity against the rice pathogen Magnaporthe oryzae. The O821-ECF significantly inhibited spore germination, appressorium formation, and mycelial growth of M. oryzae, and its antifungal activity was heat-stable. It also significantly suppressed the number and size of blast lesions. In an analysis of the ITS sequence of this isolate, it shared similarities with species of the fungus Biscogniauxia. These results suggest that isolate O821 of the genus Biscogniauxia produces a heat-stable antifungal compound(s) in its culture filtrate.     

一株疫病拮抗青霉P.st10菌株的抗菌活性及其对辣椒疫病的盆栽防效

筛选得到一株疫霉菌的拮抗青霉Penicillium striatisporumP.st10。该菌株强烈抑制疫霉菌及核盘菌的菌丝生长。无菌滤液抑制辣椒疫霉孢子囊的形成及孢子囊、游动孢子的萌发,且能杀死菌丝细胞。该菌株在辣椒根际有很强的定殖能力,有机肥对其定殖有增强作用,接种后40d在根际的青霉菌数量,青霉处理为0.76×104cfu/g,而青霉菌肥处理为2.55×104cfu/g。盆栽试验显示:处理7d后,土壤只接种辣椒疫霉对照发病率为93%;同时接种青霉培养滤液和辣椒疫霉处理为16%;接种青霉培养滤液、新鲜菌丝和辣椒疫霉处理为15%。     

Characteristics of alginate pellets formulated with Trichoderma and Gliocladium and their effect on the proliferation of the fungi in soil

Pelletized formulations of wheat bran or kaolin clay in an alginate gel containing conidia, chlamydospores, or fermentor biomass (FB) of several isolates of the biocontrol fungi Trichoderma spp. and Gliocladium virens were prepared. The ability of fungal propagules within the pellets to proliferate in soil was determined. Higher population densities were obtained when alginate pellets added to soil contained chlamydospores rather than condia, and bran rather than kaolin as the bulking agent. The active ingredient in pellets prepared from FB was approximately 5% biomass by weight and contained many chlamydospores. Colony-forming units (cfu) ranged from 10⁶'to 10¹⁰/g of soil after soil amendment with FB pellets of 12 Trichoderma and G. virens isolates. Population densities were high during the first 3 weeks of incubation and declined only gradually during 9 weeks. Propagules in FB pellets were more viable at 5° than at 25°C. Viability at 25°C remained high (> 70%) after 1 week, but declined to less than 10% after 24 weeks. Despite reduction in propagule viability in stored pellets, numbers of cfu formed after adding these pellets to soil were comparable with those formed from freshly prepared pellets.     

一种镰刀菌对空心莲子草的致病力与寄主专一性测定

从罹病的空心莲子草上分离到一种镰刀菌,人工接种条件下,3叶期的空心莲子草幼苗最易发病,叶部次之.在供试的4个菌株中,以YZ-03菌株的致病力最强,该菌株接种空心莲子草幼苗5d后,接种的植株病情指数已达100,8d后幼苗全部枯死;人工接种叶片和茎秆9d后,叶片的病情指数达90.95,接种茎秆的病情指数达85.19.采用27科61种植物对该镰刀菌进行寄主专一性测定,结果表明,该镰刀菌能使莲子草严重发病,也能侵染藜的幼苗,对包括主要农作物在内的其他59种植物均无致病性.     

薇甘菊柄锈菌生物学及其寄主专一性

在检疫温室条件下对外来入侵植物薇甘菊的寄生菌——薇甘菊柄锈菌的生物学及其寄主专化性进行了研究。结果表明，薇甘菊柄锈菌可以侵染植物叶片和叶柄等营养器官。受侵染部位起始出现褪绿斑，12～15d，自叶背产生黄色冬孢子堆，并逐步坏死和脱落，最终致寄主死亡。冬孢子黄至暗褐色，包埋在寄主组织中，无明显休眠期。高湿条件下，冬孢子易萌发产生担孢子进行再侵染。采用悬挂法对29个科、62个属的72种植物进行了寄主专化性测定，结果表明，薇甘菊柄锈菌在菊科植物天门冬、紫茎泽兰、向日葵、地胆草上形成褪绿斑，但未发现菌丝和吸器。薇甘菊柄锈菌可成功侵染薇甘菊和同属植物假泽兰。     

Impact of Fusarium oxysporum on the holoparasitic weed Phelipanche ramosa: biocontrol efficacy under field-grown conditions

Under the changing agro-climatic conditions of western Europe, the parasitic weed Phelipanche ramosa infests host crops such as tomato, hemp, tobacco and oilseed rape at an increasing rate. A Fusarium oxysporum isolate (FOG), that had effectively reduced the parasite's incidence under controlled environmental conditions, was tested in different granular formulations (pesta granules, alginate pellets) on P. ramosa parasitising tobacco under field-grown conditions. FOG reduced number and biomass of P. ramosa shoots by between 50% and 70% in three consecutive years (2006–2008). A single pesta application did not show consistent results throughout seasons; 50% reduction of P. ramosa biomass (DM) in the first year could not be repeated in the following years (20–30%). An alginate formulation applied alone performed better. However, a combination of pesta granules with alginate pellets had the highest reliable control efficacy (60–70%) of all treatments in two seasons, compared with the untreated control. Fungal population counts in soil samples did not show a close correlation to biocontrol efficacy. To understand field performance of this biocontrol agent, additional glasshouse and laboratory studies were conducted using soil from the experimental site. The glasshouse study revealed some fungistatic effects of the field soil that partly explain the reduced efficacy (-40%) in the field compared with results obtained under controlled conditions. Results show the potential of FOG for P. ramosa control. Because formulation affected the biocontrol efficacy, it may be worthwhile to test how the delivery system can be changed in order to achieve increased disease development in the field.     

进境美国苜蓿草中苜蓿黄萎病菌的检疫鉴定

从一批进境的美国苜蓿草样品中分离得到了一株与苜蓿黄萎病菌(Verticillium albo-atrum)相似的分离物M1。该分离物原始菌落为白色,边缘规则呈圆形,菌丝较致密,气生菌丝较少,苜蓿组织块不被菌丝覆盖,菌落生长速度为小于2.5 mm/d;M1在PDA上进行纯培养,前6d内,菌落为白色圆形,容易产生分子孢子轮枝状分生孢子梗,7d后菌落中央表面因产生休眠菌丝开始变成黑褐色至黑色,20d后菌落的表面和背面大部分均变黑色,仍不产生微菌核和厚垣孢子。M1的DNA用V.albo-atrum特异引物Vaa1/Vaa2进行检测,PCR扩增后得到预期330 bp的产物片段,产物序列与V.albo-atrum相应序列的相似性为100%。该分离物接种苜蓿草根部,15d后引起苜蓿黄萎病的典型症状。根据分离物的形态特征、PCR检测结果、PCR产物序列分析,以及致病性测定结果,将进境美国苜蓿草样品中的分离物M1鉴定为苜蓿黄萎病菌。     

Suppression of Dollar Spot by Hypovirulent Isolates of Sclerotinia homoeocarpa

ABSTRACT Selected hypovirulent isolates of Sclerotinia homoeocarpa were evaluated for efficacy in suppressing dollar spot of turfgrass under growth room and field conditions. Under growth room conditions, hypovirulent isolates Sh12B, Sh09B, or Sh08D of S. homoeocarpa caused 3.4 to 30.4% diseased turf in comparison to virulent isolates Sh48B and Sh14D, which caused 80.2 to 90.2% disease. In treatments that received both virulent and hypovirulent isolates, only hypovirulent isolate Sh12B significantly reduced disease as compared with the control with virulent isolates alone. In a field experiment in 1993 on swards of creeping bentgrass artificially inoculated with a virulent isolate of the pathogen, all treatments containing hypovirulent isolate Sh12B applied as a mycelial suspension, granular mix, or alginate pellets developed significantly less disease (6.3 to 20.8% diseased turf) compared with their respective formulation controls (23.8 to 31.2%). Suppression of dollar spot by treatment with mycelial suspensions of isolate Sh12B was evident up to 45 days postinoculation, and disease suppression was still significant 1 year after application when compared with the water control. Applications of hypovirulent isolate Sh09B did not reduce dollar spot in any treatments. Significant suppression of dollar spot by isolate Sh12B was also observed in the experiment conducted in 1994. In addition, suppression of dollar spot by hypovirulent isolate Sh12B was evaluated on swards with naturally occurring inoculum during 1994. Treatments with a mycelial suspension and alginate pellets of hypovirulent isolate Sh12B significantly reduced dollar spot compared with their respective formulation controls. With few exceptions, there was no statistical difference between treatments with hypovirulent isolate Sh12B and the fungicide chlorothalonil (Daconil 2787). Multiple applications of the hypovirulent isolate did not result in greater suppression of dollar spot as compared with a single application. The results indicate that hypovirulence has potential as an effective strategy for the management of dollar spot.     

Production of hydrophilic phytotoxins by <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>

We have established a reproducible strategy to purify hydrophilic phytotoxins present in the aqueous filtrate of Mycosphaerella fijiensis Morelet. The lyophilized culture filtrate is initially treated with activated charcoal, then successively purified using vacuum liquid chromatography and semipreparative high performance liquid chromatography. Phytotoxic activity was tested using a leaf-spot assay on healthy banana leaves. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.