Penetration of ovicidal fungus Verticillium chlamydosporium through the Ascaris lumbricoides egg-shells

The course of penetration of the ovicidal fungus Verticillium chlamydosporium Goddard through the Ascaris lumbricoides egg-shells was studied in electron miorographs. The contact area between the egg and the fungus is smooth at the stage of the contact of the ovicidal fungus with the egg surface, as well as at the stage of adhesion. No special attaching organs are formed which indicates that no mechanical penetration of the fungal hypha through the egg-shells is involved. Traces of enzymatical activity of the fungus penetrating upright through the egg-shells can be observed during the penetration stage proper. At the consumption stage, the branching of the ovicidal fungus becomes more dense and some hyphae grow through the egg envelopes parallelly under the egg surface. The fungus grows through all parts of the infected egg, even through its contents.     

Ultrastructure of eggs of Ascaris lumbricoides Linnaeus, 1758. I. Egg-shells

Under the light microscope the chitin-protein layer of egg-shells in ascarids appears to be a regular, hyaline and nonstructural layer of 1.5 to 2.00 microns in thickness. The outer uterine layer is usually removed during the preparation. The lipid (ascaroside) layer covers the inner surface of the chitinous layer and seems to be irregularly undulated and regularly thick over the whole surface, with the thickness up to 1 micron. In electron micrographs the fibrous structure of the lipid layer is not evident as a rule. This is probably due to washing the lipids away from this layer during the dehydration of deeper layers of egg-shells that are imperfectly fixed with glutaraldehyde. A very low permeability of the egg-shells is typical of geohelminth eggs. The layer lipid shows a distinct lamellate structure only after a prolonged fixation with osmium at higher temperature. This is supported by the studies using the method of freeze-fracturing.     

Autoradiographic study of kinetics of spermatogenesis in Ascaris lumbricoides

The kinetics of meiosis and spermiogenesis in Ascaris lumbricoides in vitro was studied by keeping 12 mature males in nutrient medium. The spermatocytes were made radioactive by supplementing the medium with 3H-thymidine for 2 h, following which they were transferred to fresh medium without 3H-TdR. The nematodes were then killed for preparing histological and squash preparations of the testis. An analysis of the migration of "hot" spermatocytes till the formation of mature spermatozoa, in Kodak AR-10 autoradiograms, suggested that the duration of meiosis and spermiogenesis was a little more than 5.5 and 7.6 days, respectively. The total duration of these two events was about 13.1 days when radioactive spermatozoa were detected for the first time.     

Visualization of chitin-protein layer formation in Ascaris lumbricoides egg-shells

The formation of chitin-protein layer in Ascaris lumbricoides egg-shells was studied using the method of chitin structures visualization by means of derivatives of stilbene-disulfonic acid (Blankophor, Bayer, FRG). Intensive formation of chitin structures in A. lumbricoides egg-shell occurred only in fertilized females in a very short portion of uterus from the site of connection of the oviduct with the uterus up to 25-30 mm distally, i.e. in the section forming about one fifth or less than one sixth of length of adult female uterus. Already 3 mm from the beginning of uterus there was a thin layer on the surface of the fertilized eggs which contained chitin detectable by Blankophor visualization, whereas the chitin-protein layer on the shells could not be demonstrated histologically. The thickness of this chitin-protein complex increased distally. At 25 mm from the beginning of uterus the chitin layer was already complete and its thickness did not increase any more.     

Nutritional requirements of the mycoparasitic fungus Verticillium biguttatum

Verticillium biguttatum was able to grow axenically in a synthetic liquid medium with a compound containing ammonium or amino group as nitrogen source, glucose as carbon source and biotin as growth factor. Among various carbon and nitrogen compounds tested, highest mycelial production was achieved with mannitol and with two ammonium salts and glutamine; sporulation reached highest values with galactose and glutamine. Highest yields of mycelium and conidia were obtained at pH 4.3 and 5.1, respectively. Although neutral and alkaline conditions were growth-limiting in the synthetic medium some growth ofV.biguttatum occurred on solid media at pH 7.0 and on sclerotia ofRhizoctonia solani in natural soil at pH 7.2–7.3.Samenvatting Een synthetisch vloeibaar medium met glucose als koolstofbron, een ammonium- of aminogroep bevattende verbinding als stikstofbron en biotine als groeifactor voldeed aan de voedingseisen vanVerticillium biguttatum. Van de verschillende koolstof- en stikstofbronnen leverden mannitol en twee ammoniumzouten en glutamine de hoogste opbrengsten aan mycelium; de opbrengst aan conidiën was het hoogst met galactose en glutamine. De hoogste opbrengsten aan mycelium en conidiën werden bereikt bij respectievelijk pH 4,3 en 5.1. Ofschoon neutrale en alkalische omstandigheden de groei vanV. biguttatum in het synthetische medium beperkten, werd enige groei vanV. biguttatum waargenomen op vaste voedingsbodems bij pH 7,0 en op sclerotia vanR. solani in natuurlijke grond bij pH 7,2–7,3.     

Morphology of the uterus of Ascaris lumbricoides in the region where fertilization and formation of egg-shell occur.

Morphology of the female reproductive system of Ascaris lumbricoides L. was studied in the region starting with the junction between the oviduct and the uterus (O-U) up to the junction of both uterine branches into the vagina with regard to the process of fertilization and formation of egg-shells. In the O-U junction morphology differed in two following sections: a continuous simple squamous up to simple cuboidal epithelium, and simple cuboidal up to columnar epithelium with broad intercellular spaces leading into the lumen of the tubular reproductive organ filled with sperm. The area in the O-U junction zone was found where the wall of the organ was formed by elongated club-shaped cells attached to the common basal lamina by a narrow pedicle. Intercellular spaces thus formed "crypts" which was covered with dilated parts of cells towards the tubular lumen. Crypts were found to be filled with sperm. This area resembles the structure known as the receptaculum seminis where the stored sperm survive. Epithelial cells of the uterus are of cuboidal up to columnar shape with signs of merocrine secretion. In the distal part of the uterus the secretory active cells probably produce viscous secreta allowing the transfer of the eggs towards the vagina. The cells of the uterus wall are elongated and because of their longer axis, they are orientated longitudinally. In centripetal parts, the cell walls do not have contact with each other and form elongated, deep furrows ("canyons") through which the sperm can run against the flow of uterus content up to the junction of the O-U, where they are stored in the spermatheca-like structure. At any time they are disposal for fertilization.     

淡紫拟青霉、厚垣轮枝菌生防菌剂对当归“麻口病”的防治效果

为了明确淡紫拟青霉、厚垣轮枝菌生防菌剂对当归生长量的影响及对麻口病的防治效果,进行了田间药效试验。结果表明,两种生防菌剂在当归移栽时拌细干土穴施对当归生长量(出苗、株高、叶片数、冠幅)有一定的影响;但可有效防治当归麻口病,其中淡紫拟青霉颗粒剂41.25kg/hm2处理组防效最高,为63.43%,与其他处理差异显著(P0.05);各处理对当归有较好的增产作用,增产率为25.78%~63.01%,其中淡紫拟青霉颗粒剂45.00kg/hm2处理增产率最高,为63.01%,其次为厚垣轮枝菌微粒剂33.75kg/hm2处理,增产率为61.67%;菌剂各处理相比对照可明显提高特等归比例。淡紫拟青霉颗粒剂41.25、45.00kg/hm2和厚垣轮枝菌微粒剂33.75kg/hm2穴施可有效防治当归麻口病,提高当归产量,可用于生产上防治当归麻口病。     

Colonization in cotton plants by a green fluorescent protein labelled strain of Verticillium dahliae

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease caused by the soil-borne fungal pathogen Verticillium dahliae. In this study, a green fluorescent protein (GFP) labelled V. dahliae strain (TV7) was obtained by transforming gfp into defoliating strain V991. Strain TV7 was used to study infection and colonization of wilt resistant cotton cultivar Zhongzhimian KV1 and susceptible cultivar 861 with the aid of confocal laser scanning microscopy. The results showed that initial infection and colonization of V. dahliae in Zhongzhimian KV1 and 861 were similar. Conidia and hyphal colonies formed and penetrated in the root meristematic and elongation zones and in the conjunction of the lateral and main roots. The invaded conidia started to germinate by 2 hpi (hours post-inoculation), penetrated into the root cortex and vascular bundles, eventually colonized in the stem xylem vessels and grew restrictedly in the individual tracheae of both resistant and susceptible cultivars. Moreover, pathogen DNA could be detected by qPCR in roots and stems of both cultivars, but its content in the wilt susceptible cultivar 861 was much higher than that in the wilt resistant cultivar Zhongzhimian KV1. The results indicated that the resistant cultivar has ability to suppress V. dahliae reproduction.     

Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae

Interactions between lettuce and a green fluorescent protein (GFP)-expressing, race 1 isolate of Verticillium dahliae, were studied to determine infection and colonization of lettuce cultivars resistant and susceptible to Verticillium wilt. The roots of lettuce seedlings were inoculated with a conidial suspension of the GFP-expressing isolate. Colonization was studied with the aid of laser scanning confocal and epi-fluorescence microscopes. Few differences in the initial infection and colonization of lateral roots were observed between resistant and susceptible cultivars. Hyphal colonies formed on root tips and within the root elongation zones by 5 days, leading to the colonization of cortical tissues and penetration of vascular elements regardless of the lettuce cultivar by 2 weeks. By 8 to 10 weeks after inoculation, vascular discoloration developed within the taproot and crown regions of susceptible cultivars well in advance of V. dahliae colonization. Actual foliar wilt coincided with the colonization of the taproot and crown areas and the eruption of mycelia into surrounding cortical tissues. Advance colonization of stems, pedicels, and inflorescence, including developing capitula and mature achenes was observed. Seedborne infection was limited to the maternal tissues of the achene, including the pappus, pericarp, integument, and endosperm; but the embryo was never compromised. Resistant lettuce cultivars remained free of disease symptoms. Furthermore, V. dahliae colonization never progressed beyond infected lateral roots of resistant cultivars. Results indicated that resistance in lettuce may lie with the plant's ability to shed infected lateral roots or to inhibit the systemic progress of the fungus through vascular tissues into the taproot.     

The Importance of Rhizosphere Interactions in the Biological Control of Plant Parasitic Nematodes—a Case Study using Verticillium chlamydosporium

Verticillium chlamydosporium has provided considerable control of root knot nematodes in a range of laboratory experiments and is one of the most promising natural enemies with potential as a biological control agent so far tested. From a detailed study of the biology and ecology of the fungus, it has been possible to identify the key factors which affect its efficacy and a strategy for its exploitation has been developed. The fungus survives in soil throughout a growing season and its concentration in soil may be increased by repeated applications. The rapid multiplication of root-knot nematodes on susceptible crops means that control must be extremely effective to prevent crop damage. If suitable methods of production and application can be developed, V. chlamydosporium, in conjunction with other methods such as the rotation of poor hosts, may provide adequate control, but the reliability of such approaches needs extensive testing. Although genetic manipulation offers the possibility of enhancing the ability of the fungus to kill nematodes, such an approach will require much research and, in the short term, the development of the fungus will rely on the exploitation of carefully selected wild types.     

Colonization of Vitis spp. wood by sGFP-transformed Phaeomoniella chlamydospora, a tracheomycotic fungus involved in Esca disease

To evaluate wood colonization and interactions with Vitis spp. of Phaeomoniella chlamydospora, a fungal agent involved in Esca disease, isolate CBS 229.95 was transformed using a pCT74 construct which contained the genetic markers for synthetic green fluorescent protein (sGFP) and hygromycin B phosphotransferase. Nine stable P. chlamydospora fungal transformants (Pch-sGFP lines) were obtained using polyethylene-glycol-mediated transformation of protoplasts. These were characterized for sgfp and hygromycin B phosphotransferase (hph) genome insertions and for sGFP fluorescence emission, using quantitative polymerase chain reaction and fluorimetric systems, respectively. No correlation was observed between sgfp copy number genome insertion and sGFP fluorescence expression. Cuttings of Vitis vinifera 'Montepulciano', 'Verdicchio', 'Sangiovese', 'Biancame', and 'Cabernet Sauvignon'; and the grapevine rootstocks 'Kober 5BB', 'SO4', '420A', '1103P', and V. rupestris were inoculated by immersion in a conidial suspension of the selected fungal Pch-sGFP71 line and incubated at 4 ± 1 and 25 ± 1°C. Wood colonization was estimated through epifluorescence microscopy and was affected by incubation temperature. After 6 months at 4 ± 1°C, the fungal growth was completely inhibited. At 25 ± 1°C, the highest extent of wood colonization was recorded in Montepulciano and Verdicchio, with the lowest in the rootstocks SO4 and V. rupestris. The expression of the Pch-sGFP71 transformed line was localized in the xylem area, primarily around the vessels. The use of sGFP-transformed P. chlamydospora helped to clarify different aspects associated with the location of this pathogen in grapevine tissue, before disease symptom expression.     

Biological control of Rhizoctonia solani on potatoes by antagonists. 1. Preliminary experiments with Verticillium biguttatum,a sclerotium-inhabiting fungus

A common mycoparasite,Verticillium biguttatum, was found to kill sclerotia ofRhizoctonia solani placed on an inert material (perlite) as well as in soil at 15°C and 20°C, but not at 10°C. Compared with the effectivity ofV. biguttatum, that ofGliocladium roseum, Gliocladium nigrovirens, Hormiactis fimicola andTrichoderma hamatum on sclerotia was only low. In laboratory experiments, treatment of sclerotia-bearing seed potatoes withV. biguttatum reduced disease symptoms in the first stage of growth of the potato plant.V. biguttatum was found to occur on the subterranean part of the potato plant. On untreated plants the surface of the sprouts was colonised byV. biguttatum originating from the soil, presumably partly in response to the presence ofR. solani mycelium. In a preliminary field experiment,Verticillium treatment did not reduce symptoms on the stem. However, there was a marked reduction in sclerotium formation on the newly formed potato tubers. This offers perspectives for a commercial use ofV. biguttatum in the control ofR. solani.     

Comparison of genotyping by sequencing and microsatellite markers for unravelling population structure in the clonal fungus Verticillium dahliae

Microsatellite genotyping of a large sample of isolates of Verticillium dahliae from diverse locations recently identified seven distinct genotypic clusters. However, these clusters were not put in the context of phenotypes known to be correlated with clonal lineages in V. dahliae. The objective of this study was to compare clusters defined by microsatellite markers with clonal lineages defined by single‐nucleotide polymorphisms (SNPs) and vegetative compatibility groups (VCGs). Genotyping isolates known to belong to specific clonal lineages (based on SNPs) with microsatellite markers determined the correspondence of clusters and lineages. All but one cluster corresponded to a known clonal lineage, allowing analysis of correlations of phenotypes with microsatellite genotypes from other studies. As shown previously, most race 1 isolates are in lineage 2A, and most isolates with the defoliating pathotype are in lineage 1A. Phylogenetic incompatibility was used to test for recombination or homoplasy caused by hypervariable microsatellite loci; incompatibility was highly correlated with the number of alleles per locus, suggesting that homoplasy caused by parallel evolution of microsatellite alleles is the cause of incompatibility. Microsatellite genotyping of lineage 1A isolates from cotton and olive in Spain over a 29‐year period revealed remarkably little variation; these markers did not mutate enough to provide insight on the spatial and temporal expansion of this clone. Overall, this study showed that microsatellite genotyping can be used to identify clonal lineages in V. dahliae, which has predictive power for inferring phenotypes of phytopathological relevance such as race and pathotype.     

Destruction of Ascaris suum eggs during their feeding to various species of beetles

Eggs of Ascaris suum (Linné, 1758) were fed to nine species of beetles of the families Scarabacidae, Carabidae and Histeridae in order to ascertain the dependence of egg penetration on the structure of mouth parts of the beetle. The mouth parts of some beetle species have various filtration apparatuses preventing the helminth eggs from penetrating into the digestive tract. Geotrupes stercorosus (Scriba) was found to possess a crushing apparatus which destroys a great number of helminth eggs penetrating into the digestive tract.     

Growth of the mycoparasitic fungus Verticillium biguttatum from different geographical origins at near-minimum temperatures

Sclerotia ofRhizoctonia solani collected from potato tubers from different countries were assayed for the presence of mycoparasites. Among the mycoparasites observedVerticillium biguttatum predominated. Its geographical distribution was not restricted to certain latitudes or soil types;V. biguttatum occurred worldwide in potato fields.The minimum growth temperature of 57V. biguttatum isolates was found to be in the narrow range from 10 to 13°C, irrespective of their geographical origin. A non-linear logistic growth model was used to describe the radial growth onRhizoctonia mycelium and nutrient agar plates. At near-minimum temperature the maximum colony radii varied considerably; they were up to 3.8 times that of the reference isolate M73. Based on parameter values for logistic growth, fast-and slow-growing isolates could be distinguished. Although the growth properties ofV. biguttatum isolates from different locations varied, the presence of fast- and slow-growing isolates was not restricted to particular areas and both types could be found in the same field. However, bioassays with selected fast- and slow-growing isolates do not support the assumption that growth at near-minimum temperatures is a relevant criterion for screening isolates ofV. biguttatum in terms of effectiveness for biological control ofR. solani.     

Suppression of clubroot and Verticillium yellows in Chinese cabbage in the field by the root endophytic fungus, Heteroconium chaetospira

Chinese cabbage seedlings inoculated with an isolate of the hyphomycete, Heteroconium chaetospira, were transplanted to the field. After 3 months, they showed a 52–97% reduction in clubroot and a 49–67% reduction in Verticillium yellows compared with noninoculated controls. H. chaetospira colonized the cortical cells, especially in the root tip region. Infected plants showed no disease symptoms. The infection process involves the formation of appressoria on the cell surface and the subsequent growth of hyphae within cells. H. chaetospira colonized 18 plant species, indicating a wide range of hosts. It may have potential as a biocontrol agent for clubroot and Verticillium yellows.     

棉花黄萎病菌提取物对棉花的抗病效果

近年来棉花黄萎病发生严重 ,重病田病株率高达 95 %以上。目前生产上缺少抗病品种 ,防治方法不健全 ,田间防治效果不理想 ,生产上急需新的防治措施。随着环境和生态问题的日益突出 ,诱导抗病性研究日益被人们重视。本研究从诱导抗病性原理出发 ,旨在探索能够有效激发、诱导棉花抗黄萎病的因子 ,并取得一些结果 ,现报道如下。1　材料与方法1 .1　供试材料　棉种为中棉 1 6 ;棉花黄萎病菌 (Verticillium dahliae Kleb)由本室分离鉴定。1 .2　激发子的制备　将棉花黄萎病菌在马铃薯葡萄糖液体培养基中 ,5℃下恒温振荡培养两周后 ,滤纸过滤 ,滤…     

植物内生细菌在棉花体内的定殖动态及对棉花黄萎病的生物防治效果

为明确植物内生细菌在棉花体内的定殖规律及对棉花黄萎病防治效果的影响,采用对峙培养法筛选对大丽轮枝菌Verticillium dahliae Kleb.抑菌活性强的植物内生细菌,通过形态及生理生化特征并结合16S r DNA和gyr B基因分析鉴定菌株;利用抗生素标记法及盆栽试验研究其在棉花体内的定殖动态及对棉花黄萎病的防治效果。结果表明:共筛选到3株对大丽轮枝菌具有强抑菌活性的菌株SZ5、DP10和CCM9,其中SZ5为蜡样芽胞杆菌Bacillus cereus,定殖在棉花根部、茎部和叶部,DP10和CCM9为枯草芽胞杆菌B.subtilis,定殖在棉花根部和茎部,3株菌在棉花根部的定殖数量均在10~3CFU/g以上;棉花苗沾根分别接入3株内生细菌10 d后再接种大丽轮枝菌孢子悬浮液,对棉花黄萎病的防治效果达79.52%~89.79%,而3株内生细菌沾根接种后立即移栽到含有大丽轮枝菌的土壤中对棉花黄萎病的防治效果为23.77%~36.03%。表明不同内生细菌菌株在棉花体内的定殖规律不同;且在棉苗中定殖一定数量后,才能对棉花黄萎病产生较高的防治效果。     

苜蓿黄萎病菌实时荧光PCR检测方法

苜蓿黄萎病菌(Verticillium albo-atrum)能够危害多种重要的农艺和园艺作物,是我国重要检疫性有害生物。本研究根据V.albo-atrum的ITS基因序列,结合张正光设计的引物Vaa-1和Vaa-2,设计一条TaqMan探针Vaa-probe,研究实时荧光PCR的检测方法,以更加快速、灵敏的检测出V.albo-atrum。利用该方法可检测到含V.albo-atrumDNA浓度0.0005 ng/μL以上的样品,大大提高了检测的灵敏度。