The Influences of Epicuticular Wax Disruption and Cutinase Resistance on Penetration of Tomatoes by Colletotrichum gloeosporioides

Conidia of Colletotrichum gloeosporioides germinated on green and ripe tomato fruit with intact epicuticular wax, and formed penetration pegs below melanized appressoria. If the delicate layer of epicuticular wax was distupted by abrasion or removed by solvents before inoculation, apparent increased diffusion of fruit substances into the inoculum stimulated fungal growth, hyphal anastomosis and the production of penetration pegs from hyaline appressoria. This was followed by cutlcle erosion centred on the penetration pegs in green fruit allowing sec-ondary growth of infection hyphae. Due to the development of cutinase resistance when the cuticle became yellow at ripening, no cuticle erosion occurred at penetrations on ripe fruit Since cuticle erosion followed penetration of the cutinase-susceptible cuticle and since penetration peg formation was not hindered by the cutinase cuticle, the process of primary penetration is regarded as mechanical.     

An unusual method of development and sporulation of Colletotrichum gloeosporioides on castor leaf – an SEM account

Development and sporogenesis of Colletotrichum gloeosporioides on castor leaf differed from that on other known host plants. C. gloeosporioides had three kinds of hyphae on castor leaf: primary infection hyphae (PIH), runner hyphae (RH) and secondary infection hyphae (SIH). The PIH originated from conidia, grew on leaf surface and entered the leaf by direct penetration of the cuticle without forming appressoria. The RH were sub-cuticular hyphae, the track of which was traceable by the bulgings on the leaf surface, and the SIH were the hyphae that emerged to leaf surface from RH through the cuticle or stomata. Conidia were initiated as small protrusions along the lengths of RH and SIH that got differentiated into distinct conidia, each born on a short stumpy conidiophore without forming any congregation. The protrusions from RH emerged to the leaf surface by piercing the cuticle, and they developed into distinct conidia on the leaf surface. The conidia developed from RH and SIH were identical in size and shape. Even though conidia were occasionally found emerged through stomata, that appeared to be random than a preferred route for the discharge of conidia. The penetration and sporogenesis of C. gloeosporioides on castor leaf differed from that reported on mulberry leaf.     

Antifungal effects of dimethyl trisulfide against Colletotrichum gloeosporioides infection on mango

Colletotrichum gloeosporioides, one of the main agents of mango anthracnose, causes latent infections in unripe mango and can lead to huge losses during fruit storage and transport. Dimethyl trisulfide (DMTS) is an antifungal agent produced by several microorganisms or plants, but its effects on the infection process of C. gloeosporioides have not been well characterized. A histological investigation demonstrated that DMTS exhibits strong inhibitory effects on the infection process of C. gloeosporioides in planta by inhibiting the germination of conidia and formation of appressoria, damaging cytoplasm to cause cells to vacuolate and contributing to deformation of appressoria prior to penetration. This is the first study to demonstrate antifungal activity of DMTS against C. gloeosporioides on mango by suppression of the infection process, thus providing a novel postharvest biorational control for mango anthracnose.     

Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

Abstract

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.     

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.     

Histological and Ultrastructural Changes in Leaves and Stems of Resistant and Susceptible Chickpea Cultivars to Ascochyta rabiei

The histo- and cytopathological effects in resistant (ILC-195) and susceptible (Canitez-87) chickpea cultivars were examined by light, transmission and scanning electron microscopy 3, 5 and 7 days after inoculation (d.a.i) of seedlings with Ascochyta rabiei. The fungus produced typical appressoria that penetrated both cuticle and stomata. The resistant plants had physical barriers and a cuticle layer against fungal penetration 3 d.a.i. The fungus spread intercellularly and subepidermally in the leaves and stems of susceptible plants 3 d.a.i., and was followed 5 d.a.i. by cell plasmolysis, degeneration of organelles and of cellulose, but not lignified, walls. Pycnidia formation occurred between 5 and 7 d.a.i. 7 d.a.i., organelle degeneration, pycnidia formation and symptom severity increased. Tracheidal elements, including lignified elements, were almost intact in both resistant and susceptible cultivars. In the susceptible plants, lignin cell walls were slightly degraded after 7 days. There was less cell degeneration and pycnidia formation in resistant plants. Some electron-dense large bodies and lipid granules were observed within intracellular fungal hyphae in infected cells of resistant plants 7 d.a.i.     

球孢白僵菌不同感染方式侵染棉铃虫幼虫的毒性比较及组织病理变化

为明确球孢白僵菌在不同感染方式下对棉铃虫的侵染能力, 采用饲喂法和浸渍法测定了球孢白僵菌HFW-05对棉铃虫Helicoverpa armigera (Hübner) 2龄幼虫的致病力, 并通过组织切片显微技术、 扫描电镜技术观察了球孢白僵菌HFW-05对棉铃虫的致病方式。结果表明: 白僵菌HFW-05可通过消化道（饲喂法）成功侵染2龄棉铃虫, 接种感染6 d后的校正死亡率为75.8%。经由体表（浸渍法）接种白僵菌HFW-05的试虫, 试验中体重的变化和取食量与对照相近（6 d校正死亡率仅为17.3%, 不能通过体表达到致病效果）。组织病理学变化表明: 26±1℃条件下, HFW-05菌株对棉铃虫以消化道侵染为主, 侵染后可导致寄主中肠微绒毛脱落严重, 肠壁组织溶解并最终只剩余底膜; 马氏管变形萎缩, 边缘向外突出隆起, 管径变大; 脂肪体萎缩解体, 结构松散; 表皮下的细胞被菌丝侵染破坏。浸渍法接种的试虫, 切片观察处理6 d后试虫, 体内未发现菌丝, 肠壁组织正常完整。扫描电镜观察, 浸渍法接种的分生孢子未能穿透棉铃虫表皮, 而是贴于寄主表皮表面生长, 在湿度合适的条件下, 菌丝生长到一定时间后断裂成为芽生孢子。白僵菌HFW-05可经由消化道对棉铃虫达到较高的致病效果, 在一定程度上弥补外界环境对白僵菌侵染的不利影响, 对今后应用白僵菌进行生物防治具有重要意义。     

Surface ultrastructure of the uredinial stage ofCerotelium fici and its infection process on mulberry

Surface morphology of uredinia and urediniospores ofCerotelium fici (Cast.) Arth., and its infection process in mulberry (Morus alba L.) have been described using the scanning electron microscope. The uredinia ofC. fici are paraphysate and bear pedicellate urediniospores. The surface morphology of urediniospore is similar to most of the rust fungi which have pedicellate urediniospores. The infection process ofC. fici on mulberry leaves differs from other rust fungi in not forming appressoria over the stomates. Further, the germ tube of the urediniospore crosses over the stomata, and sometimes forms an appressorium close to the stoma rather than forming over it. Thus, the present study indicates that the formation of appressoria byC. fici on mulberry leaves is not site specific but an independent, specialized and inherent mechanism required byC. fici to penetrate the mulberry leaf cuticle and epidermis.     

Histology of Disease Development in Resistant and Susceptible Cultivars of Chickpea (Cicer arietinum L.) Inoculated with Spores of Ascochyta rabiei

Abstract Histological studies were performed on a compatible and an incompatible interaction between chickpea ( Cicer arietinum L.) plants and the fungus Ascochyta rabiei (Pass.) Labr. The time course of infection, development on leaflets and stems of susceptible (ILC 1929) and resistant (ILC 3279) plants was monitored by light or scanning electron microscopy with the aim to compare histological changes as the basis for further work on biochemical changes in this plant-pathogen interaction.
Spores of A. rabiei began to germinate from 12 hpi on and developed a polar germ tube; fungal colonization, secretion of a mucilaginous exudate and appressoria formation (1–3 dpi) were identical on both cultivars. Leaves of susceptible plants were invaded by the fungus directly through the cuticle, the fungus then spread subepidermally followed by a rapid collapse of the leaf tissue (4–6 dpi). Development of leaf spots and fungal pycnidia could be observed 6–8 dpi. The resistant cultivarrapidly responded (24–48 hpi) to fungal infection and cells of the palisade parenchyma exhibited autofluorescence. In later stages of the infection (4–5 dpi) fluorescent areas developed to small necrotic spots all over the leaflet. These necrotic areas, were the result of cell death and a subsequent change in the leaf structure and were characterized by the accumulation of phenolic compounds. Leaves of the resistant cultivar were invaded by the fungus to less than 5%.     

The biology of Peronospora viciae on pea: factors affecting the susceptibility of plants to local infection and systemic colonization

Peronospora viciae (Berk.) Casp. penetrated leaf disks of Pisum sativum L. through the cuticle. Resistance of pea plants and of individual leaves to infection by P. viciae increased with age, but decreased again at senescence. Resistance was shown by a restriction in fungal growth and sporulation and by a chlorotic reaction in the leaves. Systemic invasion followed infection of meristematic tissue, and was induced by inoculation into the apical bud of young plants, or on to the epicotyl or hypocotyl, but not roots of germinating seedlings. Most plants whose growth was retarded showed an increased resistance to systemic infection. Pods were infected externally by sporangia, rather than by mycelial growth through the peduncle and pedicel. Oospores and mycelium were found in the testas of some seeds, but seeds from infected pods did not give rise to infected seedlings.     

The role of cuticle and epidermal cell wall in resistance of rapeseed and mustard to Rhizoctonia solani

A study was conducted to determine whether the cuticles in two genera of the family Cruciferae are effective barriers to infection by Rhizoctonia solani, and whether differences in cuticle and epidermal cell wall thickness and morphology of epicuticular wax exist between resistant and susceptible cultivars. As Canola/rapeseed (Brassica napus) and mustard (Sinapis alba) plants develop from 1 to 3 weeks of age, they become increasingly resistant to R. solani AG2-1 seedling root rot. Seven-day-old seedlings of S. alba cultivars are invariably more resistant than B. napus cultivars. Brassica napus cultivars do not show an obvious cuticle layer at 1 week but at 3 weeks the presence of a cuticle is seen through autofluorescence with a concomitant increase in resistance to R. solani. Removal of the cuticle from 3-week-old hypocotyls by chloroform treatment results in a decrease in cuticular autofluorescence and a significant increase in disease severity in both resistant and susceptible cultivars. Three-week-old plants of S. alba have a much lower percent disease rating and a significantly (p=0.05) thicker cuticle layer than similar-age plants of B. napus. The results suggest that the cuticle plays an important role in the resistance of S. alba and older plants of B. napus to infection by R. solani.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

Prepenetration and Penetration of Colletotrichum gloeosporioides into Guava Fruit (Psidium guajava L.): Effects of Temperature,Wetness Period and Fruit Age

In this study, we determined the influences of temperature, wetness period and guava fruit age on infection caused by Colletotrichum gloeosporioides. Optimal temperatures in vitro for germination, appressoria formation and melanization were 22.7, 20.6 and 23°C, respectively. In vivo, the optimal temperatures for germination and appressoria formation were 22.5 and 23°C, respectively. Values for germination, appressoria formation and melanization were higher as the wetness period increased. There was no difference in conidial germination and appressorial formation on fruit of different ages. On the surface of 10‐, 35‐ and 60‐day‐old fruit, despite the high percentage of appressorial formation, there was no development of the penetration peg. Penetration pegs were only observed on the 85‐ and 110‐day‐old fruit. Thickness of the cuticle, size and architecture of epidermal and parenchymal cells, as well as the content of phenolic compounds changed as the fruit ripened.     

Fungi growing on stored soybeans and their significance in lipid breakdown*

US Grade 2 soybeans contain about 20% cracked and split seeds, 3% damaged seeds and 2% foreign material. Examination by scanning electron microscopy showed that intact seeds were externally almost free of microorganisms. In cracked seed coats and in damaged areas on broken soybeans, profusely developing fungal mycelia were frequently observed. Fungi penetrated the beans through these cracks and through the micropyle. Penetration through the hilum was observed only in cases where the seed coats showed pronounced signs of damage. No penetration through pore-like structures in the seed coat could be observed. Light microscope observations of sections of naturally infested soybeans showed little penetration into the underlying cell layers. Sections of soybeans kept at 99% r.h. and 26d?C for 4 days revealed profusely developing fungi colonising both seed coat tissue and cotyledons. In the cotyledonous tissue hyphae did not penetrate into the cells but developed only in the intercellular spaces. In inoculated, detached seed coats, fungi developed better on the inner than on the outer side. On cotyledons free of seed coats, fungal development was better than in the seed coat itself. In broken soybeans of commercial stock origin, free fatty acids (FFA), mould count and respiration rate increased 3.5-, 3.5-and 5.0-fold, respectively as compared with intact ones. When broken and intact soybeans were mixed there was no increase in the rate of deterioration. It is concluded that fungi are involved in the increase of FFA in soybeans during storage and that this process is significantly faster in the broken bean fraction.     

Ultrastructure of Tobacco Ringspot Virus-Induced Local Lesions in Lima Bean Leaves

Lignin is hypothesized to be a factor in the ability of cucurbits to resist fungal infection. To determine if lignin was present around sites of infection, susceptible cucumber plants and cucumber plants with systemic induced resistance were inoculated with Colletotrichum lagenarium, and the ultrastructure of plant and pathogen was examined 24 to 72 hours later. Conidia produced germ tubes with appressoria within 24 hours of inoculation on both control and induced plants. Penetration of plant epidermal cell walls occurred by 48 hours, and papillae formed at the site of penetration. Both elemental bromine and potassium permanganate were used to histochemically stain for lignin. Potassium permanganate, used together with energy dispersive X-ray microanalysis (EDS), indicated the presence of lignin in papillae and in electron-dense areas of plant cell walls directly beneath the appressoria. Using EDS, silicon was shown to be localized in the electron-dense areas of cucumber leaf cells. This is the first report of silicon in induced cucumber plants. There were no qualitative differences between the resistance reactions of induced and control plants.     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Transpiration inhibition by stored xylem sap from well-watered maize plants

There is increasing evidence that a chemical signal exists in xylem sap of plants subjected to water deficits which influences physiological responses in plant shoots. An important method of studying this signal is the transpiration response of excised leaves exposed to xylem sap collected from plants. However, Munns et al [Plant, Cell & Environment 16, 867–877] cautioned that transpiration inhibition is observed when xylem sap collected from wheat and barley is stored before determining physiological activity. The objective of the study reported here was to determine if transpiration inhibition develops in maize sap collected from well-watered plants when the sap is stored under various conditions. It was found that storage of maize sap collected from well-watered plants for only 1 d at -20°C resulted in the development of substantial transpiration inhibition in bioassay leaves. Storage of sap at 4°C resulted in the development of the effect after 2 weeks, while storage at ?86°C showed only small transpiration inhibition after 3 weeks. The major source of the transpiration inhibition was the development of a substance in the stored sap that resulted in physical blockage of the transpiration stream in bioassay leaves. However, a small signal component may also have developed in the stored sap. Because of the possibility of ionic activity under freezing conditions at ?20°C, calcium was studied for its potential involvement in the transpiration inhibition. However, the calcium concentrations found to inhibit transpiration were nearly an order of magnitude larger than the calcium concentrations observed in xylem sap.     

Comparative headspace analysis of cabbage plants damaged by two species ofPieris caterpillars: consequences for in-flight host location byCotesia parasitoids

Headspace composition, collected from intact cabbage plants and cabbage plants infested with eitherPieris brassicae L. orP. rapae L. (Lepidoptera: Pieridae) first instar larvae, was determined by GC-MS. Twenty-one volatiles were identified in the headspace of intact plants. Twenty-two volatiles were identified in the headspace of plants infested byP. brassicae larvae, 2 of which, Z-3-hexenyl butyrate and Z-3-hexenyl isovalerate, were not detected in the headspace of either intact orP. rapae damaged plants. In the headspace of the latter, 21 compounds were identified, all of which which were also produced by intact plants. No significant quantitative differences were found between headspace composition of the plants damaged by one or the other caterpillar species. Major differences between intact and caterpillar-damaged plants in contribution to the headspace profile were revealed for hexyl acetate, Z-3-hexenyl acetate, myrcene, sabinene and 1,8-cineole. The larval endoparasitoidCotesia glomerata L. was attracted by the volatiles emanating fromB. oleracea damaged byP. brassicae first instar larvae.C. rubecula L., a specialized larval endoparasitoids ofP. rapae, was attracted by the volatiles released from theB. oleracea-P. rapae plant-host complex. This shows that cabbage plants kept under the conditions of headspace collection produce attractive volatiles for both parasitoids.     

Infection of sunflower leaves by ascospores of Sclerotinia sclerotiomm

Ascospores of Sclerotinia sclerotiorum infected fully expanded sunflower leaves in the absence of added nutrients, wounds or senescent tissue. The site of infection was confined to a specific region around the junction of the leaf blade and the petiole and was associated with sites of sucrose secretion by the host. In these areas ascospores germinated and formed extensive colonies on the leaf surface. Simple appressoria were visible at 24 h, and complex appressoria at 48 h, after inoculation. Ascospores that germinated in areas where sucrose was not secreted produced germtubes that were shorter (less than 50 μm at 48 h after inoculation) than those produced at sites where sucrose was secreted (greater than 5 mm in length). Infections also developed on severely wounded main leaf veins. Leaf wetness was required for infection, with maximal infection occurring after a 72 h dew period.