Identification of hydrolytic activities expressed by Aspergillus flavus grown on cotton carpel tissue

To identify proteins important for invasion into host plant tissue, Aspergillus flavus was cultured on medium containing cotton carpel tissue as the sole carbon source. We identified several hydrolases suggesting they are important as A. flavus virulence factors for plant colonization. Specifically, Aspergillus flavus AF13 secreted at least two endoxylanase activities and a pectolytic activity when grown on the cotton carpel tissue medium. A concentrated sample derived from the A. flavus growth medium (6-day) was subjected to gel filtration chromatography on a BioGel P-30 column. A major endoxylanase activity was separated from the other fungal-secreted proteins. Additional fungal secreted proteins were partially resolved by gel filtration chromatography on a BioGel P-60 column. Multiple proteins with molecular weights in the 20 to 70 kD range were present in the harvested fungal growth medium. Analysis of these fungal-secreted proteins by liquid chromatography/tandem mass spectrometry identified both endo- and exo-glucanase proteins, α-l-arabinofuranosidase, glucoamylase, α-amylase A, pectate lyase A, xylanase F1, acetylxylan esterase, glutaminase A, as well as conserved hypothetical proteins of unknown function. These proteins likely assist A. flavus in the maceration of plant cell walls, allowing for pathogenic entry and accession of host nutrient resources. Pectolytic and xylanolytic hydrolases, as well as glucanases, appear to be important A. flavus virulence factors.     

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.     

Use of some plant extracts to control Biomphalaria alexandrina snails with emphasis on some biological effects

The present work was carried out to evaluate, the molluscicidal activity of cold water, boiled water, methanol, ethanol, acetone and chloroform extracts of some plant species against Biomphalaria alexandrina snails. Preliminary screening tests on 10 plant species showed that the highest molluscicidal potency was recorded for Euphorbia splendens, Atriplex stylosa and Guayacum officinalis. Exposure of B. alexandrina snails to plant’s methanol extracts led to a significant reduction in their survival and growth rates. In addition, newly hatched snails were susceptible to plant’s methanol extracts than elder ones. LC₂₅ of methanol extract from these plants caused a considerable reduction in the infectivity of Schistosoma mansoni miracidia to the snails. It caused a reduction in number of cercariae per snail during the patent period and in the period of cercarial shedding. The results, also, revealed that the glucose concentration in treated snails was increased in haemolymph, while soft tissue glycogen decreased. The activities of glycogen phosphorylase, succinate dehydrogenase (SDH), glucose-6-phosphatase and acetylcholinesterase (AchE) in homogenate of snail’s tissues were reduced (P < 0.001) in response to treatment with plants methanol extract, while glucose-6-phosphate dehydrogenase (G-6-PD) activity increased (P < 0.001). It was concluded that the application of LC₂₅ of methanol extracts of E. splendens, A. stylosa and G. officinalis may be helpful in snail control as it interferes with the snails’ biology and physiology.     

Induction of hypericins and hyperforin in Hypericum perforatum L. in response to biotic and chemical elicitors

Hypericum perforatum L. produces hyperforins, a family of antimicrobial acylphloroglucinols; and hypericins, a family of phototoxic anthraquinones exhibiting anti-microbial, anti-viral, and anti-herbivore properties in vitro. To determine whether these secondary metabolites are part of the specific plant defense systems that are mediated by methyl jasmonate or salicylic acid, we used meristem cultures to assess the effects of exposure to exogenous application of these chemical elicitors. Levels of hypericins in plant tissue increased in response to both elicitor treatments; total hypericin levels increased as much as 3.3 times control levels when treated with 200 μ methyl jasmonate for 14 days. Increased hyperforin concentrations were detected when plantlets were treated with 1 m salicylic acid or 50 μ methyl jasmonate. For assessing responses to a biotic elicitor, greenhouse-grown plant materials were inoculated with the plant pathogen, Colletotrichum gloeosporioides. Levels of hypericins increased twice as much as the control when inoculated with 1 × 10⁴ spores per ml; higher doses of spores overwhelmed the plant defenses. The elevation of hypericins and hyperforin in response to chemical and biotic elicitors suggests that these secondary metabolites are components in the inducible plant defense responses of H. perforatum.     

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.     

Detection of the phytotoxin coronatine by ELISA and localization in infected plant tissue

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR)in vitro, and the availability of purified toxin has facilitated development of immunological detection methods. A modified, indirect competitive ELISA using the COR-specific monoclonal antibody 11B8 was developed to detect COR in various host plants infected by P. syringae. The estimated detection limit for COR was 50 pg per well, and COR could be reliably quantified from 5 to 40 ng ml⁻¹. The subcellular localization of COR within infected tomato tissue was investigated using the COR-specific antibody MAb 8H3G2. Immunofluorescence microscopy and immunogold labelling showed that COR was present inside tomato cells and was associated with chloroplasts and particles of proteinase inhibitor I. Localization studies indicated that COR is mobile in infected plant tissue and can be detected in healthy tissue adjacent to the bacterial lesions.     

Defense strategies of pea embryo axes with different levels of sucrose to Fusarium oxysporum and Ascochyta pisi

The aim of this study was to compare the defense responses of embryo axes of Pisum sativum L. cv. Kwestor with different sucrose levels to pathogenic fungi, i.e. systemic acting Fusarium oxysporum f. sp. pisi and locally acting Ascochyta pisi. Embryo axes were cultured on Heller medium for 96 h. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). After inoculation of the pea embryo axes with pathogenic fungi a generally higher concentration of free radicals was detected by electron paramagnetic resonance (EPR), in comparison to non-inoculated embryo axes. The inoculation with F. oxysporum caused stronger generation of free radicals in −Si than in +Si embryo axes. A different response was observed after inoculation with A. pisi; starting from 48 h, the concentration of free radicals in +Si axes was found to be 1.5 times higher than in −Si embryo axes. The values of spectroscopic splitting coefficients for these radicals suggest that they are semiquinone radicals. The EPR method also revealed Mn²⁺ ion accumulation after 24 h of culture. Over time, high levels of these ions were recorded in +Si embryo axes inoculated with F. oxysporum, while in +Si embryo axes inoculated with A. pisi they decreased. Up to 48 h after inoculation with the pathogenic fungi, Mn²⁺ ion levels were higher in +Si embryo axes than in +Sn axes. The activity of superoxide dismutase (SOD, EC 1.15.1.1) increased in +Si embryo axes up to 72 h after inoculation with pathogenic fungi; however, it was generally lower than in +Sn axes. Catalase activity (CAT, EC 1.11.1.6) increased up to 72 h after inoculation with F. oxysporum and the values were higher than in the non-inoculated tissue. Especially high activity of this enzyme was noted in −Si embryo axes after inoculation with either F. oxysporum or A. pisi. Peroxidase activity (POX, EC 1.11.1.7) towards pyrogallol in embryo axes increased during culture; however, it was lower or similar to that in non-inoculated embryo axes. SOD, CAT and POX zymograms showed that the synthesis of new isoforms was induced after inoculation with pathogenic fungi. Peroxidase isozymes detected by the reaction with diaminobenzidine in native PAGE were intensely stained in +Si embryo axes after inoculation with pathogenic fungi. Respiratory activity of the inoculated tissues was considerably higher than in non-inoculated tissues. The respiration rate was generally much higher in +Si than in −Si embryo axes. Growth of −Si embryo axes was more significantly retarded as a consequence of inoculation than that of +Si embryo axes.These results indicate that, depending on the manner of influence of a pathogenic fungus, both similar and differing defensive strategies may be initiated and a raised sugar levels in pea tissues limit the development of F. oxysporum and A. pisi.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

Reinforcement of cell wall in roots of Lycopersicon esculentum through induction of phenolic compounds and lignin by elicitors

Changes in phenolic metabolism and lignin deposition have been studied in roots of tomato plants after elicitation with four elicitors which are Fusarium mycelium extract (FME), chitosan (CHT), Fusarium culture filtrate (FCF) and Trichoderma mycelium extract (TME). Most profound effect of elicitors was observed on ferulic acid among the phenolic compounds. After 24 h elicitation, the increase in ferulic acid content of root cell wall was 3.71 and 3.30 times by FME and CHT, respectively. The increase of 4-hydroxybenzoic acid was 2.71 and 2.16 times by these two elicitors. The level of 4-coumaric acid was little more than double by these two elicitors after 24 h elicitation. Most pronounced increase in lignin synthesis was also effected by FME followed by CHT. Lignin deposition in the root cell wall was increased 3.6, 5.4 and 7.1 times by FME during 12, 24 and 36 h after elicitation, respectively. Similarly, CHT increased lignin deposition by 2.8, 5.1 and 6.8 times at 12, 24 and 36 h after elicitation, respectively. FCF and TME also increased lignin deposition significantly in the cell walls of tomato roots during the above time periods of elicitation. Activity of phenylalanine ammonia lyase reached highest level at 24 h post elicitation under the influence of the elicitors. Peroxidase activity registered a sharp increase at 24 h post elicitation. Markedly increased level of polyphenol oxidase activity was found at 12 h post elicitation. Cinnamyl alcohol dehydrogenase activity was observed to reach highest level at 48 h post elicitation. Cell wall strengthening, through the deposition of lignin, preceded by the induction of the synthesizing enzymes appears to play an important role in the defense response of Lycopersicon esculentum in reaction to elicitors, including one derived from Fusarium oxysporum f. sp. lycopersici, the causal organism of Fusarium wilt of tomato.     

Correlation of ethylene synthesis in Citrus fruits and their susceptibility to Alternaria alternata pv. citri

The susceptibility of Fortune (Citrus clementina × Citrus reticulata), Citrus paradisi and Citrus limon fruits to Alternaria alternata pv. citri was investigated using different artificial inoculation methods. The results obtained reveal that the C. paradisi and C. limon fruits are less susceptible to A. alternata pv. citri than Fortune fruits, although all showed symptoms of Alternaria brown spot when the cuticle was broken and the flavedo or flavedo + albedo was removed. Furthermore, it was seen that susceptibility to the fungus decreased as the age of the fruit increased. There was a positive correlation between the susceptibility of the different Citrus fruits to A. alternata pv. citri and their “in vivo” ethylene levels, the most susceptible fruit (Fortune) producing more ethylene during growth than the less susceptible C. limon and C. paradisi. This suggests that ethylene may well be considered as a possible marker of Citrus fruit susceptibility to A. alternata pv. citri. Disease development increased when the Fortune fruits were treated with 1 mM ACC (a precursor of ethylene biosynthesis) or 1 mM Ethephon (an ethylene-releasing compound) prior to inoculation with A. alternata pv. citri. The role of ethylene as a factor involved in disease development is discussed.     

Effects of inoculum density,leaf wetness duration and nitrate concentration on the occurrence of lettuce leaf spot

In Ehime Prefecture, Japan, lettuce leaf spot (Septoria lactucae) caused huge losses in marketable lettuce yields. To explore potential measures to control disease outbreaks, the effects of inoculum density, leaf wetness duration and nitrate concentration on the development of leaf spot on lettuce (Lactuca sativa) were evaluated. Conidia were collected from diseased plants in an infested field by single-spore isolation and were used to inoculate potted lettuce plants with different conidial concentrations. Lesions developed on inoculated lettuce plants at inoculum concentrations from 10⁰ to 10⁶ conidia/ml. The disease was more severe when the inoculum exceeded 10² conidia/ml, and severity increased with increasing concentrations. Assessment of the relationship between disease development and the duration of postinoculation leaf wetness revealed that symptoms appeared when the inoculated plants remained wet for 12 h or longer. The number of lesions and total nitrogen content in the lettuce leaves both increased when nitrate was applied.     

Gel detection of Allium porrum polygalacturonase-inhibiting protein reveals a high number of isoforms

Polygalacturonase inhibiting proteins (PGIPs) are leucine-rich repeat glycoproteins, localized in the cell wall of most plant species, capable of countering the activity of endo-polygalacturonases (endo-PGs) produced by phytopathogenic fungi. The PGIP from Allium porrum leaves was analysed to ascertain the presence of different molecular forms of PGIP. Leek PGIP was separated into two fractions: a soluble and an ionically wall-bound PGIP, each of which was then purified by cation-exchange chromatography. Two and three peaks of PGIP activity were obtained, respectively. PGIP isoforms contained in each peak were separated by isoelectrofocusing (IEF) on a polyacrylamide gel. Following the separation, the gel was first overlaid with sodium polygalacturonate and then treated with the endo-PG from either Sclerotinia sclerotiorum, Fusarium moniliforme or Botrytis aclada. The endo-PG(s) hydrolyse the overlaid substrate except where active inhibitors are present. The presence of PGIPs is revealed by ruthenium red staining of the nonhydrolysed substrate. Each PGIP peak following IEF separation revealed several PGIP isoforms with pIs between 5.0 and 7.0. More than 20 isoforms were detected in total, with considerable differences in their inhibitory activity. While similar PGIP isoform patterns were obtained by developing the IEF gels with the endo-PGs of S. sclerotiorum and B. aclada, less intense PGIP bands were observed with the endo-PG from B. aclada, consistent with inhibition assays performed in solution. The endo-PG from F. moniliforme, which is not inhibited at all by leek PGIP in solution, consistently showed no PGIP band on the gel assay.     

黄淮海夏玉米主产区穗腐病病原菌的分离鉴定

为明确我国黄淮海夏玉米主产区玉米穗腐病的病原菌种类、优势种群及虫害、年度、省份对病原菌的影响,以形态学为基础,结合分子生物学方法对2013、2015年随机采自河南、河北、山东3省的155份玉米穗腐病样品进行分离鉴定。结果表明,引起黄淮海夏玉米主产区玉米穗腐病的主要致病菌为镰孢菌Fusarium spp.,包括拟轮枝镰孢F.verticillioides、禾谷镰孢F.graminearum、层出镰孢F.proliferatum、木贼镰孢F.equiseti及藤仓镰孢F.fujikuroi,分离频率分别为49.7%、28.4%、12.3%、3.9%和1.3%;其次为木霉菌Trichoderma spp.,包括哈茨木霉T.harzianum、绿色木霉T.viride和棘孢木霉T.asperellum,分离频率分别为8.4%、3.2%和5.2%;青霉菌Penicillium spp.分离频率较低,为14.2%;曲霉菌Aspergillus spp.包括黑曲霉A.niger和黄曲霉A.flavus,分离频率分别为2.6%和1.9%。研究表明,黄淮海主产区玉米穗腐病优势病原菌为拟轮枝镰孢、禾谷镰孢和木霉菌,不同省份不同年度间病原菌种类及优势病原菌存在差异,虫害能加重玉米穗腐病的发生。     

Spatial and temporal changes in acetolactate synthase activity as affected by new herbicide ZJ0273 in rapeseed, barley and water chickweed

Propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273), derived from a precursor compound pesticide (a derivative of 2-pyrimidinyloxy-N-aryl benzoate), is a novel herbicide used in the rapeseed field. This paper studied the differential tolerance or susceptibility of four plant species viz. Brassica napus L. (rapeseed), Hordeum vulgare L. (barley), Malachium aquaticum (L.) Fries (water chickweed), and Alopecurus aequalis Sobol. (equal alopecurus), to various concentrations of ZJ0273, based on the rate of inhibition of acetolactate synthase (ALS) activity. Brassica napus was observed as the most tolerant among four species in which ALS activity in vivo was hardly affected by ZJ0273 at 100 mg/L (recommended dose in the rapeseed field). In contrast, H. vulgare was found to be more susceptible which was reflected in the relative decline of ALS activity and lower biomass production even under at a lower rate of 10 mg/L of ZJ0273. The two weed species exhibited moderate susceptibility as compared to H. vulgare. The present study confirmed that ZJ0273 at 100 mg/L is the optimal dose of herbicide for rapeseed field. Seedling stage was critical in determining the tolerance of crops and susceptibility of weeds which indicated that over seven leaf stage was much safer for rapeseed whereas the two leaf stage juvenile water chickweed was more susceptible to the same rate of ZJ0273. Spatial and temporal variations in ALS activity as affected by 100 mg/L ZJ0273 in water chickweed, rapeseed and barley also suggested that the synchronized sampling based on the tissue position and seedling stage was necessary to examine precisely, the special difference in tolerance or susceptibility to ALS-inhibiting herbicide. The uppermost younger leaf seemed to be the most appropriate sample for evaluation. Furthermore, the variant tendency of ALS activity from different parts of plants between monocotyledons and dicotyledons to ZJ0273 treatments showed differential response which revealed some possible mechanism related to the altered absorption and translocation of the herbicide.     

喜旱莲子草次生代谢物对莲草直胸跳甲谷胱甘肽S-转移酶和淀粉酶活性的影响

为明确喜旱莲子草的次生代谢物对其专食性天敌-莲草直胸跳甲Agasicles hygrophila成虫解毒酶和消化酶的影响,利用不同浓度的橙花叔醇、齐墩果酸和甜菜碱浸叶处理喜旱莲子草Alternanthera philoxeroides,连续饲喂莲草直胸跳甲1~3 d后,测定莲草直胸跳甲谷胱甘肽S-转移酶(GSTs)和淀粉酶(AMS)的活性变化。结果表明,橙花叔醇、齐墩果酸和甜菜碱均能抑制莲草直胸跳甲的GSTs活性,0.1%橙花叔醇和20%甜菜碱处理24 h时的GSTs活性最低,分别为45.67 U/mg和53.95 U/mg,0.2%齐墩果处理72 h时的GSTs活性最低,为98.77 U/mg,且莲草直胸跳甲对橙花叔醇的刺激存在着一种适应机制;0.5%橙花叔醇和0.1%齐墩果酸处理24 h后的GSTs活性最高,分别为243.10 U/mg和250.22 U/mg,是对照的1.55倍和1.59倍,存在诱导激活现象。甜菜碱对莲草直胸跳甲的AMS活性有明显的抑制作用,20%甜菜碱处理72 h后AMS活性最低,为1.01 U/mg,是对照的55.92%。表明喜旱莲子草3种次生代谢物均能抑制谷胱甘肽S-转移酶活性,其中橙花叔醇和齐墩果酸对谷胱甘肽S-转移酶有诱导激活作用,而甜菜碱对淀粉酶抑制作用较强。     

Detection of viridiofungin A and other antifungal metabolites excreted by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> active against different plant pathogens

Antibiosis is assumed to be an essential mechanism exerted by potential biocontrol agents (BCAs) of Trichoderma spp. Therefore, in the present study, we report for the first time on the elucidation and production of viridiofungin A (VFA) from T. harzianum isolate T23 cultures and investigate the antifungal potential of VFA and some other secondary metabolites purified from T. harzianum cultures against Fusarium moniliforme. The bioautography assay revealed that T. harzianum isolates T16 and T23 excreted several secondary metabolites with antifungal activity. Following isolation and purification of the antifungal zones, three fractions (F223, F323 and F423) from extracts of isolate T23 and two fractions (F416 and F516) from extracts of isolate T16 exhibited pronounced fungitoxic activity in the bioautography and antibiotic disk assays against Cladosporium spp. and F. moniliforme, respectively. The structure of the antifungal metabolite in fraction F323 was identified as viridiofungin A (VFA), the first report of production of VFA by isolate T23 of T. harzianum. Following cultivation of isolate T23 in PDB medium for 9 days, 94.6 mg l⁻¹ of VFA were determined. VFA and fraction F516 retarded the mycelial growth of F. moniliforme in the non-volatile phase assay by >90% for each 250 μg ml⁻¹ 7 days post-inoculation (dpi). While VFA and fraction F416 showed both volatile and non-volatile effects, fraction F516 seemed to exhibit mainly non-volatile activity. Microscopic examination revealed that hyphae of F. moniliforme grown on VFA-amended medium were less branched and appeared thicker than untreated hyphae. Furthermore, in the presence of VFA, formation of chlamydospores by F. moniliforme was increased. Finally, the antifungal spectrum of VFA towards various important plant pathogens was evaluated. Germination of propagules of a variety of fungal pathogens in vitro was differentially inhibited by VFA. While in the presence of 100 μg ml⁻¹ VFA conidial germination of V. dahliae was completely inhibited, a slightly higher concentration (150 μg ml⁻¹) of the inhibitor was required to suppress germination of Phytophthora infestans sporangia or sclerotia of Sclerotinia sclerotiorum. Contrary to several reports in the literature, VFA proved to be fungistatic rather than fungicidal. However, neither VFA nor the other Trichoderma metabolites, such as 6PAP, F416 and F516, exhibited any antibacterial activity against Gram-positive and Gram-negative bacteria.     

植物内生枯草芽孢杆菌Em7菌株对葡萄灰霉病菌的抑菌活性

通过室内皿内对峙抑菌试验、分生孢子萌发抑制试验、离体果实接种试验以及电镜技术,研究测定了分离自小麦根部的植物内生枯草芽孢杆菌Em7菌液对葡萄灰霉病菌Botrytiscinerea Pers.的抑制作用及抑菌机理。结果表明:用Em7菌液处理葡萄灰霉病菌后,在PDA培养基上形成了明显的抑菌圈,直径达2.81 cm;菌液对分生孢子萌发的抑制率达到88.65%;经Em7菌液处理后,离体果实病情指数明显低于空白对照,相对防治效果达到78.92%。电镜观察发现,处理组菌丝生长异常,体表凹凸不平,局部膨大成结或缢缩,分枝变多,菌丝体内液泡增多,细胞壁增厚,细胞膜透性发生变化。表明植物内生枯草芽孢杆菌Em7菌株对葡萄灰霉病菌有良好的抑制作用,并且可以有效控制葡萄灰霉病的发生。     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

Influence of plant host species on intraspecific competition during infection by Aspergillus flavus

Compositions of Aspergillus flavus populations determine the extent to which crops become contaminated with aflatoxins. In the current study, influences of diverse crop hosts on competition among A. flavus isolates were quantified with pyrosequencing. Maize, cotton, soyabean and sorghum supported different levels of sporulation, but intraspecific differences in sporulation were not detected on any host. However, hosts differentially influenced competition during infection, allowing greater sporulation by some isolates and increased host tissue invasion by others. Furthermore, competitive interactions during host invasion did not predict isolate success during sporulation. Isolates were similarly competitive on maize and sorghum, the two most closely related hosts. Host‐specific influences on intraspecific competition may dictate compositions of A. flavus populations and, as a result, the severity of aflatoxin contamination. Host factors should be considered when designing and implementing aflatoxin management strategies including biocontrol with atoxigenic strains.     

Infection of wheat spikes by <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> and alterations of cell wall components in the infected tissue

The infection process of Fusarium avenaceum on wheat spikes and the alteration of cell wall components in the infected host tissue were examined by means of electron microscopy and cytochemical labelling techniques following spray inoculation at growth stage (GS) 65 (mid-flowering). Macroconidia of the pathogen germinated with one to several germ-tubes 6–12 h after inoculation (hai) on host surfaces. The germ-tubes did not penetrate host tissues immediately, but extended and branched on the host surfaces. Hyphal growth on abaxial surfaces of the glume, lemma and palea was scanty 3–4 days after inoculation (dai) and no direct penetration of the outer surfaces of the spikelet was observed. Dense mycelial networks formed on the inner surfaces of the glume, lemma, palea and ovary 36–48 hai. Penetration of the host tissue occurred 36 hai by infection hyphae only on the adaxial surfaces of the glume, lemma, palea and upper part of ovary. The fungus penetrated the cuticle and hyphae extended subcuticularly or between the epidermal wall layers. The subcuticular growth phase was followed by penetration of the epidermal wall, and hyphae spread rapidly inter- and intracellularly in the glume, lemma, palea and ovary. During this necrotrophic colonization phase of the wheat spike, a series of alterations occurred in the host tissues, such as degeneration of cytoplasm and cell organelles, collapse of host cells and disintegration of host cell walls. Immunogold labelling techniques showed that cell walls of spike tissues contained reduced amounts of cellulose, xylan and pectin near intercellular hyphae or infection pegs compared to walls of healthy host tissues. These studies suggest that cell wall degrading enzymes produced by F. avenaceum facilitated rapid colonization of wheat spikes. The different penetration properties of abaxial and adaxial surfaces of the spikelet tissues as well as the two distinct colonization strategies of host tissues by F. avenaceum are discussed. The penetration and colonization behaviour of F. avenaceum in wheat spikelets resembled that of F. culmorum and F. graminearum, although mycotoxins produced by F. avenaceum differed from those of the latter two Fusarium species.