荸荠茎点霉秆枯病菌侵染过程的超微观察

<正>荸荠(Eleocharis dulcis),又称马蹄,为莎草科多年生草本植物,是一种具有食用和药用价值的水生蔬菜。近年来,随着荸荠在我国种植面积的不断扩大,病害发生也呈逐年上升趋势。荸荠茎点霉秆枯病是2009年在湖北省荸荠产区发现的一种新病害,由Phoma bellidis侵染引起,该病在湖北省团风地区发生尤为严重,对荸荠的产量和品质造成严重影响;病害一般在8~12月发生,发病初期在荸荠茎秆上产生圆形或梭形红褐色小斑,随后病斑沿茎     

Nutritional factors affecting the behaviour of Uromyces viciae-fabae uredospores on broad bean leaves

Both germination of Uromyces viciae-fabae uredospores and growth of germ tubes were increased when spores were leached for 12 h at 0.4 ml/min water flow whereas leaching at aflow rate of 2.8 ml/min completely inhibited germination. Washing uredospores once in water caused a reduction in growth of germ tubes and in the numbers of lesions produced on detached leaves of the host, broad bean (Vicia faba).
During germination uredospores from recently formed pustules leaked considerably moreendogenous nutrients on hydration than older uredospores. However, the majority of the leaked compounds were reabsorbed by young uredospores within a few hours. Concentrations ofglucose and leucine equivalents leaked from uredospores and from leaves were compared.
Leaf leachates stimulated growth of uredospore germ tubes to an extent similar to that caused by low concentrations of glucose and amino acids but the addition of pollen inhibited growth of germ tubes. A variety of nutrients caused stimulation of uredospore germination and growth of germ tubes at low concentrations whereas higher concentrations of exogenous nutrients inhibited these processes. The response of uredospores was affected by their age.
Utilization of exogenous ¹⁴C-labelled nutrients by germinating uredospores was very low compared with that of cells of a leaf surface yeast, Cryptococcus sp. Cryptococcus ; cells could consume over 90% of the glucose in a solution of glucose and amino acids of concentrations similar to those in leaf leachates before germinating U. viciae-fabae uredospores had taken up 10%.     

Responses of Erysiphe graminis germlings to contact with artificial and host surfaces

Germling development by Erysiphe graminis f. sp. hordei was compared between conidia held in a simulated air-borne state on microthreads constructed from safety-line threads produced by orb--weaving spiders (Araneus diadematus), and conidia inoculated onto glass, agar, or living or dead barley coleoptile epidermes. Suspended conidia germinated but generally produced only multiple short germ tubes. Conidia on living or dead coleoptiles, bathed from beneath with 0.01 Ca(NO₃)₂ solution, generally produced one short germ tube and a second germ tube which elongated and formed a normal appressorium. On glass and agar, multiple short germ tubes were sometimes formed but long germ tubes were formed less frequently than on host epidermis. When conidia with short germ tubes were transferred from microthreads to coleoptiles, they produced a long germ tube which differentiated an appressorium. Conidia with a single short germ tube were also transferred from microthreads so that only the tip of the short germ tube was in contact with a leaf epidermal strip layed on agar, whilst the conidium rested on the agar. Long germ tubes were formed more frequently by such conidia than by controls which had no contact with the leaf epidermis. This suggested that a stimulus causing elongation of the second tube was perceived through the short germ tube in contact with the epidermal strip. Where long germ tubes made contact with the epidermal strip, normal appressoria were formed more frequently than where the long tube made contact with the agar surface alone. The results indicate that germlings develop through distinct stages in response to particular stimuli.     

青杨叶锈病菌(Melampsora larici-populina Kleb.)侵染过程的超微结构研究

 采用电子显微镜技术对青杨叶锈病菌(Melampsora larici-populina Kleb.)的侵染过程进行了研究。发现该菌夏孢子萌发产生1~3个芽管,且具较多的树杈状分枝。芽管由气孔侵入,侵入前不形成明显的附着胞或仅个别芽管形成附着胞。芽管侵入气孔后在气孔腔内形成气孔下囊,再分化出圆形的膨大体而产生1~2支初生菌丝。初生菌丝在寄主细胞间扩展,与叶肉细胞壁接触后分化出吸器母细胞,吸器母细胞中的细胞器与胞间菌丝相同,双核。吸器母细胞产生侵入钉侵入叶肉细胞内部形成吸器,成熟吸器由细长具颈环的管状颈部和膨大的吸器体组成,此时胞间菌丝在吸器母细胞处分化出次生菌丝,在叶肉细胞间扩展形成次生菌落,产生孢子堆。病菌在寄主细胞间隙或沿寄主细胞壁延伸时,寄主细胞仍保持正常状态。     

苹果炭疽叶枯病病原Glomerella cingulata及其侵染过程

为明确苹果炭疽叶枯病病原菌围小丛壳Glomerella cingulata的侵染致病特征,在分离获得该病原菌的基础上,采用形态学观察、ITS序列分析和致病性测定对其进行了鉴定,并利用光学和扫描电子显微镜对病原菌在嘎啦苹果叶片上的侵染过程进行了研究.结果表明,在陕西咸阳地区分离获得的9株病原菌均为围小丛壳G.cingulata.25 ℃下接种9 h后,分生孢子中间产生隔膜,双胞化,并萌发产生芽管和附着胞;24 h后分生孢子的2个细胞均可萌发并形成芽管及附着胞,部分芽管顶端可产生次级分生孢子;48 h后次级分生孢子萌发形成附着胞;72 h后,附着胞下形成的侵染钉可直接入侵寄主,在表皮细胞内形成初生菌丝和次生菌丝,此时叶片表面已出现褐色斑点.接种7 d后叶片病斑处出现分生孢子盘和子囊壳.表明陕西省近年出现的苹果炭疽叶枯病病原菌为围小丛壳G.cingulata,该病菌在嘎啦叶片上的一些特殊侵染行为可能是导致该病害易在短时间内暴发的重要原因.     

Host specialization of <Emphasis Type="Italic">Tranzschelia discolor</Emphasis> on stone fruits at the uredinial infection stage

The host range specialization of Tranzschelia discolor (Fckl.) Tranz. & Litv. on stone fruits at the uredinial infection stage was investigated. Aeciospore inocula were collected from the alternate host, Anemone coronaria, at ten different locations of Aydın Province in early spring and were separately inoculated to 1–2-year-old nursery stocks of plum, peach, apricot, almond and sweet cherry under growth chamber conditions. Rust pustules developed only on plums, 15–21 days after inoculations, but no infections occurred on the other stone fruits. Urediniospore inocula were collected from naturally infected plums, apricots, peaches and almonds and were inoculated to four host species as well as sweet cherry in a series of cross-inoculations. Rust pustules developed on plants of the respective original host species from which the urediniospore inoculum was collected. No morphological differences were found among teliospores and urediniospores of T. discolor sampled from the different stone fruit species. Results of this study indicate that there is host specialization of T. discolor on stone fruits at the uredinial infection stage. Aecial inoculum from anemones in Aydin Province was infectious only on plums. The alternate host for the other stone fruits in this province was not found.     

The role of primary germ tubes in the life cycle of Blumeria graminis: The primary germ tube is responsible for the suppression of resistance induction of a host plant cell

When the conidia of Blumeria graminis f. sp. hordei (Bgh) are inoculated on barley coleoptile cells they produce short germ tubes called primary germ tubes (PGTs) about 2 h after inoculation. We evaluated the positive role of a PGT in inducing accessibility of the host cell under the germ tube. When an appressorium (APP) penetrated the same cell on which a PGT was present, the ratio of haustorium formation (penetration efficiency) was significantly higher than when an APP penetrated the cell adjacent to the one on which a PGT was present. When an APP penetrated the cell laterally adjacent to the one on which a PGT was present we killed the cell under the PGT by puncturing it with a microneedle and then investigated the penetration efficiency of the cell adjacent to the dead cell. As a control we killed the cell longitudinally adjacent to the one on which a PGT was present and investigated the penetration efficiency of the laterally adjacent cell. The results showed that the penetration efficiency of the former was significantly lower than that of the latter. This suggests that some accessibility factor might transfer from a cell on which a PGT is present to a laterally adjacent cell. The existence of a conidium body but not a PGT was not effective for induced accessibility of the host cell. Moreover, when a Bgh germling was removed 6 h after inoculation and another germling was transferred to the same cell, the penetration efficiency was significantly higher than that of control. As a control, a Bgh germling was transferred to a cell on which no germling was present. These results suggest that the existence of PGT is effective for induced accessibility of a host cell when penetrated by Bgh. However, it is unclear whether or not a PGT secretes some substance(s) which suppresses the resistance induction of a host cell.     

The role of primary germ tubes in the life cycle of Blumeria graminis: The primary germ tube is responsible for the suppression of resistance induction of a host plant cell

When the conidia of Blumeria graminis f. sp. hordei (Bgh) are inoculated on barley coleoptile cells they produce short germ tubes called primary germ tubes (PGTs) about 2 h after inoculation. We evaluated the positive role of a PGT in inducing accessibility of the host cell under the germ tube. When an appressorium (APP) penetrated the same cell on which a PGT was present, the ratio of haustorium formation (penetration efficiency) was significantly higher than when an APP penetrated the cell adjacent to the one on which a PGT was present. When an APP penetrated the cell laterally adjacent to the one on which a PGT was present we killed the cell under the PGT by puncturing it with a microneedle and then investigated the penetration efficiency of the cell adjacent to the dead cell. As a control we killed the cell longitudinally adjacent to the one on which a PGT was present and investigated the penetration efficiency of the laterally adjacent cell. The results showed that the penetration efficiency of the former was significantly lower than that of the latter. This suggests that some accessibility factor might transfer from a cell on which a PGT is present to a laterally adjacent cell. The existence of a conidium body but not a PGT was not effective for induced accessibility of the host cell. Moreover, when a Bgh germling was removed 6 h after inoculation and another germling was transferred to the same cell, the penetration efficiency was significantly higher than that of control. As a control, a Bgh germling was transferred to a cell on which no germling was present. These results suggest that the existence of PGT is effective for induced accessibility of a host cell when penetrated by Bgh. However, it is unclear whether or not a PGT secretes some substance(s) which suppresses the resistance induction of a host cell.     

The rapid and accurate determination of germ tube emergence site by Blumeria graminis conidia

The development of appressoria by germinating Blumeria graminis conidia depends on its germ tubes making contact with the host surface. Low angle, low temperature scanning electron microscopy showed that 80% or more of first-formed germ tubes of f. spp. hordei, tritici and avenae conidia emerged from close to the host leaf surface, and so made contact with it allowing them to become functional primary germ tubes. Light microscopy of f. sp.hordei confirmed this result and, in addition, showed that germ tubes frequently emerged close to, and contacted, various hydrophobic and hydrophilic artificial substrata. Geometric models of conidium-substratum interfaces were developed and a “surface point method” was derived to predict the frequency with which contact would result if germ tube emergence was a random phenomenon. However, observed contact frequencies were far higher (c. three to eight times) than predicted. Thus, the germ tube emergence site was determined as a response to substratum contact. In part, this appeared to be a non-specific response. Nevertheless, germ tube contact frequencies were greater on the curved surface of leaf epidermal cells than on planar surface, suggesting that specific recognition of leaf surface characteristics may promote directional emergence. The area of contact required to stimulate directional germ tube emergence was very small: contact with a microneedle tip or with a spiders’ suspension thread was sufficient for many conidia. Similarly, on leaves, the only contact is between the tips of a limited number of conidial wall projections and the edges of epicuticular leaf wax plates. Micromanipulation to roll conidia so that their original site of contact with a leaf was rotated away from it, led to the majority of first-formed germ tubes growing away from the leaf, i.e. emerging close to the site of original contact. The experiments indicated that the site of germ tube emergence is determined within 1 min of deposition. This implicated the release of conidial extracellular materials in recognition of the conidium-leaf surface contact site.     

Variation in the pine stem rust fungus Peridermium pini in the United Kingdom

Peridermium pini aeciospores from lesions on Scots pine ( Pinus sylvestris ) in Scotland and in Thetford Forest, East Anglia, differed in terms of germ tube morphology and cytology. The former produced long aseptate germ tubes containing a pair of nuclei. The latter produced short septate germ tubes, each cell containing a single nucleus. One third of the 'Scottish' germ tubes ended in a vesicle. Nuclei could be counted in only a small proportion of ungerminated aeciospores. On average 83% of Scottish spores had two nuclei and 4% had one. The equivalent figures for Thetford spores were 66 and 20%. A small number of aeciospore samples from other parts of England resembled Scottish spores rather than Thetford spores in germ tube morphology and cytology.
Both Scottish and Thetford spores proved capable of causing infection of wounded Scots pine shoots. In addition, some Thetford spores caused infection on leaf discs taken from certain Paeonia cultivars. From these results it is concluded that two distinct forms of Peridermium pini exist in the UK. The implications of this finding are discussed in relation to the history of pine stem rust in the country and the genetics and taxonomy of P. pini.     

小麦矮化腥黑穗病菌侵染能力的研究

中美专家合作在美国犹他州对小麦矮化腥黑穗病菌的侵染能力进行了研究。试验采用不含抗病基因的小麦品种Cheyanne,播种前用溴甲烷对试验小区进行了熏蒸。接种物为含有多种致病型的冬孢子悬浮液,采用土壤表面喷洒冬孢子悬浮液的方法进行接种,接种密度为每cm20、0.88、8.84、88.45、884.49、8844.89、88449个孢子。结果表明,接种密度为每cm20.88个孢子时即可产生0.21％的穗发病率。对试验数据进行分析后表明,小麦矮化腥黑穗病菌的侵染规律符合侵染概率理论,在本试验条件下,单个孢子的侵染概率为0.0002。     

唐松草作为小麦叶锈菌的转主寄主问题的研究

1984年在内蒙古卓资县白石头沟野外调查中发现4种唐松草和5种禾本科杂草都发生有叶锈病。但无迹象表明该地区自然界唐松草上发生的叶锈病与小麦叶锈病有关。在室内用小麦、华北剪股颖和无芒雀麦叶锈菌的冬孢子对亚欧和瓣蕊唐松草接种,都长出了锈孢子腔和锈孢子。用三种锈孢子分别对小麦、华北剪股颖和无芒雀麦回接,都产生了夏孢子,完成了生活循环。从此得出结论:亚欧和瓣蕊唐松草是小麦、叶锈菌的转主寄主,但在室外不起作用。而田间所见唐松草上的锈病主要与杂草上的锈病有关。     

唐松草作为小麦叶锈菌的转主寄主问题的研究

 1984年在内蒙古卓资县白石头沟野外调查中发现4种唐松草和5种禾本科杂草都发生有叶锈病。但无迹象表明该地区自然界唐松草上发生的叶锈病与小麦叶锈病有关。在室内用小麦、华北剪股颖和无芒雀麦叶锈菌的冬孢子对亚欧和瓣蕊唐松草接种,都长出了锈孢子腔和锈孢子。用三种锈孢子分别对小麦、华北剪股颖和无芒雀麦回接,都产生了夏孢子,完成了生活循环。从此得出结论:亚欧和瓣蕊唐松草是小麦、叶锈菌的转主寄主,但在室外不起作用。而田间所见唐松草上的锈病主要与杂草上的锈病有关。     

Disease cycle of Austropuccinia psidii on Eucalyptus globulus and Eucalyptus obliqua leaves of different rust response phenotypes

Myrtle rust poses a significant biosecurity threat to Australia with potential for long-term damaging impacts on native flora and plant industries. This study describes the disease cycle of Austropuccinia psidii, the myrtle rust pathogen, in Eucalyptus globulus and Eucalyptus obliqua, two commercially and ecologically important species from different subgenera of Eucalyptus. Ontogeny and morphology of infection structures of A. psidii on plants of both Eucalyptus species with different rust response phenotypes, i.e. completely resistant, hypersensitive and highly susceptible, were investigated. Plants were inoculated with single-uredinium-derived urediniospores and examined by scanning electron microscopy. No differences between rust response phenotypes were observed in germination of urediniospores, formation of appressoria or length of germ tubes. The growth of germ tubes had no affinity towards stomata of either species. Histological observations indicated direct penetration by infection pegs through the leaf cuticle and no penetration beyond the epidermis on rust-resistant E. obliqua. Eucalyptus obliqua plants that were identified as susceptible to A. psidii at 3- and 6-months-old showed no disease when reinoculated with A. psidii at 12-months-old; this indicated possible early acquisition of adult plant resistance to A. psidii in this species. In the susceptible phenotype of E. globulus rust inoculation led to rapid colonization of leaf parenchyma cells with the disease cycle completed within 10 days. These findings provide valuable insights into host–pathogen interactions in the Eucalyptus–A. psidii pathosystem, which might be useful for the development of effective rust control strategies across Eucalyptus subgenera.     

Use of Green Fluorescent Protein-Transgenic Strains to Study Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum

ABSTRACT Colletotrichum acutatum, which causes anthracnose disease on strawberry, can also persist on several other plant species without causing disease symptoms. The genetic and molecular bases that determine pathogenic and nonpathogenic lifestyles in C. acutatum are unclear. We developed a transformation system for C. acutatum by electroporation of germinating conidia, and transgenic isolates that express the green fluorescent protein (GFP) were produced. Details of the pathogenic and nonpathogenic lifestyles of C. acutatum were determined by using GFP-transgenic isolates. Major differences between colonization-mediating processes of strawberry and of other plants were observed. On the main host, strawberry, the germinating conidia formed branched, thick hyphae, and large numbers of appressoria were produced that were essential for plant penetration. In strawberry, the fungus developed rapidly, filling the mesophyll with dense mycelium that invaded the cells and caused necrosis of the tissue. In nonpathogenic interactions on pepper, eggplant, and tomato, the conidia germinated, producing thin, straight germ tubes. Appressoria were produced but failed to germinate and penetrate leaf tissue, resulting in epiphytic growth without invasion of the plant. Penetration of the plant occurred only several days after inoculation and was restricted to the intercellular spaces of the first cell layers of infected tissue without causing any visible damage. Much of the new fungal biomass continued to develop on the surface of inoculated organs in the nonpathogenic interaction. The differences in fungal development on strawberry compared with the other plant species suggest that signal molecules, which may be present only in strawberry, trigger appressorial germination and penetration of the primary host.     

Pollen mimicry during infection of blueberry flowers by conidia of Monilinia vaccinii-corymbosi

Infection of blueberry flowers by Monilinia vaccinii-corymbosi, which causes mummy berry disease, occurs via the gynoecium and involves conidial germination on the stigmatic surface followed by hyphal ingress into the stylar canal and subsequent colonization of the ovary. The extent to which these events mimic pollen–pistil interactions during pollination was investigated. Similar to blueberry pollen tubes, conidial germ tubes of M. vaccinii-corymbosi adhered selectively to imprints of stylar transmitting tract tissue on nitrocellulose membrane, with adhesion in both cases occurring at the tips of the tubes. By contrast, hyphae of the related Monilinia fructicola, which is nonpathogenic on blueberry and does not cause gynoecial infection, adhered indiscriminately to the entire membrane. Using monoclonal antibodies, the presence of epitopes of esterified and unesterified pectins and of arabinogalactan proteins (AGPs), which have been implicated in adhesion and pollen tube guidance in other plant species, was documented on blueberry pollen tubes in vitro. Epitopes of certain AGPs, but not of pectins, were also localized on conidia and hyphae of M. vaccinii-corymbosi. However, such epitopes were also detected on M. fructicola, suggesting that they are unlikely to be a discriminatory factor between fungi capable or not capable of gynoecial infection. Microscopic observation of inoculated pistils showed that similar to pollen tubes, hyphae of M. vaccinii-corymbosi tracked the lobes of the stylar lumen, grew directionally (i.e. with very limited branching) in close proximity to cells of the stylar adaxial epidermis and to one another, and were surrounded by extracellular matrix. By contrast, hyphae of M. fructicola, while being able to ingress into the style, branched profusely within the stylar canal, showing no directional growth or affinity to specific regions of the lumen. We propose these results as evidence of specialized opportunism by M. vaccinii-corymbosi, whereby fungal hyphae appear to mimic host pollen tubes and take advantage of an infrastructure intended to support host reproduction in order to facilitate infection of the ovary.     

Strawberry Anthracnose: Histopathology of Colletotrichum acutatum and C. fragariae

ABSTRACT Ontogeny of the invasion process by Colletotrichum acutatum and C. fragariae was studied on petioles and stolons of the strawberry cultivar Chandler using light and electron microscopy. The invasion of host tissue by each fungal species was similar; however, each invasion event occurred more rapidly with C. fragariae than with C. acutatum. Following cuticular penetration via an appressorium, subsequent steps of invasion involved hyphal growth within the cuticle and within the cell walls of epidermal, subepidermal, and subtending cells. Both species of fungi began invasion with a brief biotrophic phase before entering an extended necrotrophic phase. Acervuli formed once the cortical tissue had been moderately disrupted and began with the development of a stroma just beneath the outer periclinal epidermal walls. Acervuli erupted through the cuticle and released conidia. Invasion of the vascular tissue typically occurred after acervulus maturation and remained minimal. Chitin distribution in walls of C. fragariae was visualized with gold-labeled wheat germ agglutinin. The outer layer of bilayered walls of conidia, germ tubes, and appressoria contained less chitin than unilayered hyphae in planta.     

Anastomosis of germ tubes and migration of nuclei in germ tube networks of the soybean rust pathogen, <Emphasis Type="BoldItalic">Phakopsora pachyrhizi</Emphasis>

An important mechanism for genetic diversity in filamentous fungi is hyphal anastomosis and the formation of heterokaryons. In this study, we observed fusion of germ tubes in germinating urediniospores of Phakopsora pachyrhizi resulting in a complex hyphal network. Staining of the germ tubes derived from P. pachyrhizi urediniospores with 4’, 6-diamidino-2-phenylindole (DAPI) showed migration of nuclei through the network resulting in multinucleate hyphae. Short bridges connecting the hyphal network tubes were also observed. Our study provides the first evidence of germ tube and hyphal anastomosis, and nuclear migration in P. pachyrhizi. Considering the lack of a known sexual stage of P. pachyrhizi, this hyphal anastomosis followed by the parasexual cycle may explain the genetic diversity in virulence among populations of P. pachyrhizi.     

Microscopical studies of the infection of gerbara flowers byBotrytis cinerea

The formation of lesions on ray florets of gerbera flowers caused by single conidia ofBotrytis cinerea was studied in two cultivars infected by two isolates of the pathogen. No differences in reaction after inoculation with conidia of either isolate were seen on either cultivar. The conidia produced usually one germ tube not longer than 10 m, but conidia with five germ tubes were also seen. Direct penetration of germ tubes through the upper cuticle of ray florets was observed. No appressoria or other specialised structures were observed before penetration, and degradation of the cuticle did not occur. Germination of conidia and subsequent flower infection was dependent on the availability of free water, but not on the addition of external nutrients.Between 18 to 25°C, fungal development usually stopped after cuticle penetration, two to four cells around the site of penetration becoming necrotic. This number did not increase when inoculated flowers were subsequently placed at 4°C, conditions conductive for the formation of spreading lesions. When flowers were incubated constantly at 4°C, lesions became visible 3 days after inoculation as a group of 10 to 14 cells. Initially from a vesicle-like structure, mycelium spread subcuticularly or in the lumen of epidermal cells resulting in the death of 40 to 50 cells at 18 days after inoculation. Ungerminated conidia and conidial germlings which has not yet penetrated the cuticle did not cause any visible symptoms in underlying epidermal cells.     

Evaluation of screening protocols to determine genetic variability of grain sorghum germplasm to Sporisorium sorghi under field and greenhouse conditions

Covered kernel smut caused by Sporisorium sorghi is a serious problem in grain sorghum (Sorghum bicolor)-growing areas in the world, particularly where seed dressings are unavailable or too expensive. In a screening protocol designed to eliminate the possibility of escapes, seed subjected to a teliospore suspension coupled with a partial vacuum, and seeds directly infested with dried teliospores, were equal in development of smutted sorghum panicles. Inoculation with sporidia from teliospores was ineffective when plants were inoculated at the boot stage of growth and was minimally effective when plants were inoculated at the 10-12 leaf stage of growth. Environmental conditions were more conducive to disease incidence when sorghum was grown under greenhouse conditions or planted late in the field. Sorghum planted in warm (>20C) soil had the greatest incidence of covered kernel smutted panicles.