Visualization of localized pathogen-Induced pH modulation in almond tissues infected by Colletotrichum acutatum using confocal scanning laser microscopy

Modulation of pH within the host during infection of almond by the anthracnose pathogen Colletotrichum acutatum was studied using confocal scanning laser microscopy and the dual emission fluorescence indicator SNARF-1. This highly sensitive method allowed visualization of the spatial distribution of localized pathogen-induced pH modulation within and in proximity to fungal infection structures in host tissue at the cellular level. Ratiometric measurement of fluorescence at two emission wavelengths and in situ calibration allowed the quantification of pH ranges. After incubation of leaf epidermal tissue with SNARF-1, distinct alkaline (pH 8 to > or =9), red-spectrum (650 nm wave length) fluorescent zones developed as partial or complete halos around many fungal appressoria and in infection vesicles at 24 to 36 h after inoculation. In samples taken after 48 to 72 h, colonizing hyphae in the biotrophic phase and subsequently in the necrotrophic phase were also emitting the red fluorescence that extended into the surrounding host tissue, as also verified by depth analyses. Host epidermal cells were intact and apparently alive during the fungal alkalization process, with no visible disruption of cell structure. Generally, the pH of epidermal cells in noninoculated samples or in areas away from the infection in inoculated samples was lower than pH 7 with green (i.e., 500 to 550 nm wave length) fluorescence detected. Using standard electrodes, a significant increase in pH and ammonia concentration in leaf and fruit tissue was also measured but only at advanced stages of disease. In contrast, hyphae of the pathogen Alternaria alternata were mostly acidic and no change in fluorescence was found inside invaded host cells. The sequence of events in the C. acutatum-almond interaction includes penetration, production of ammonia by C. acutatum, and subsequent pH modulation within almond epidermal tissue to an alkaline environment that leads to further colonization of the host.     

The Effect of Nitrogen on Disease Development and Gene Expression in Bacterial and Fungal Plant Pathogens

Successful colonisation of plants by pathogens requires efficient utilisation of nutrient resources available in host tissues. Several bacterial and fungal genes are specifically induced during pathogenesis and under nitrogen-limiting conditions in vitro. This suggests that a nitrogen-limiting environment may be one of the cues for disease symptom development during growth of the pathogens in planta. Here we review recent literature on the effect of nitrogen and nitrogen-regulated genes on disease development, caused by phytopathogenic bacteria and fungi. Furthermore, the potential influence of nitrogen-limitation or general nutrient limitation on several in planta-induced bacterial and fungal pathogenicity, virulence and avirulence genes will be discussed.     

猕猴桃采后病害鉴定和侵染规律研究

 猕猴桃果实在0℃贮藏4周后开始出现腐烂,贮藏19周,腐烂率高达50%以上。腐烂部位几乎都在果蒂和花端两点以外的果身上,极少数是蒂腐和花端腐烂。病原菌有7种,其中Botrytis cinerea,Penicillium sp.和Phomopsis sp.3种为优势菌。病原菌主要通过采收运输时造成的机械伤口侵染,未发现在果实过熟衰老前直接侵染。     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

Pathogen behaviour and plant cell reactions in interactions between Alternaria species and leaves of host and nonhost plants

The growth behaviour of several Alternaria species on leaf discs of host and nonhost plants was examined by u.v. microscopy. Specific patterns of development were found for the pathogens. Differences in development which correlated to host specificity, or which differentiated specialized pathogens from weak or opportunistic pathogens, were obvious only after attempted penetration. Thus, little variation was observed amongst different plants for individual Alternaria species in the rate of germination, in the extent of germ-tube growth or in the frequency of appressorium production. However, plant responses to attempted penetration, which included the formation of callose-containing papillae, callose deposition in the walls of attacked cells and their neighbours and cell necrosis, varied with specific pathogen–plant interactions. Callose deposition occurred at sites of both successful and unsuccessful penetrations and may, therefore, not be a determining factor in the plant–pathogen interactions examined. A biplot technique is used to illustrate the different degrees of host specificity apparent at pre- and post-penetration stages of fungal development.     

Infection of plum and nectarine flowers by Botrytis cinerea

The ability of Botrytis cinerea to infect plum and nectarine flowers was studied in moist chambers and in the orchard. Within 36 h of inoculation, the pathogen penetrated and colonized the stamens, styles and petals on shoots placed in moist chambers, causing blossom blight. Similar lesion development was observed following inoculation with dry and wet conidia, Hyphae were usually distorted in stylar tissue, but grew normally in petals and filaments. Growth of the fungus through filaments into the sepals or floral tubes, or through the transmitting tissue of the style into the ovary, was never observed. Symptoms of blossom blight were not observed on inoculated shoots in the orchard. The floral tube, bearing the sepals and stamens, dehisced within 14 days of fruit set and infected floral parts did not remain attached to young developing fruit. No relation was found between post-harvest decay and flower infection. Losses following post-harvest decay might have been caused by direct penetration of ripening fruit and not by flower infection. The importance of infected floral parts as a source of secondary inoculum on ripening fruit is discussed.     

Modulation of host pH during the wheat–Fusarium culmorum interaction and its influence on the production and activity of pectolytic enzymes

The effect on ambient pH of Fusarium culmorum during its growth on mineral medium and in apoplastic fluids from infected wheat seedlings, and the effect on the production and activity of the enzymes pectin lyase (PNL) and polygalacturonase (PG), were investigated. Fungal development on a weakly buffered mineral medium in the pH range 5·0–8·0, with pectin as the sole carbon source, led to pronounced alkalinization, reaching values above 8·0. The increase in ambient pH was accompanied by enhancement of total PNL activity. Pectin lyase secretion was detected at pH 5·0 as a single isoenzyme. An additional isoenzyme was apparent during the increase in medium pH. Polygalacturonase was detected as a single isoenzyme only during early growth on medium buffered at pH 5·0. At this stage, the initial medium pH of 5·0, corresponding to the optimum pH for PG activity, appeared to be the most suitable for the activation of early production of this enzyme. During growth in acidified yeast extract medium the fungus secreted ammonia and increased medium pH. Similarly, in apoplastic fluids from inoculated seedlings the concomitant ammonia accumulation and rise in pH were recorded. This trend was accompanied by an increase in PNL, which could therefore function at close to its optimal pH. The results suggest that during infection of wheat seedlings by F. culmorum , pH modulation can lead to PNL production and activity, thus promoting colonization of host tissue.     

The relative importance of Colletotrichum musae as a crown-rot pathogen on Windward Island bananas

Low numbers of conidia of Colletotrichum musae (10–50) applied directly to the surface of freshly cut banana crowns caused extensive rot development. At least 20 times more conidia of Fusarium pallidoroseum were required to cause comparable levels of crown rot. Examination of the relative pathogenicity of the major fungal species involved in development of crown rot, C. musae, F. pallidoroseum, Fusarium moniliforme, F. moniliforme var. subglutinans , and Botryodiplodia theobromae , using a standard inoculum of 2 × 10³ conidia per crown, showed C. musae to be the most aggressive species. C. musae produced a distinctive soft, dry fibrous rot while the fruit was still green, and on ripening further rotting, softening and blackening of crown tissues occurred. The other crown-rot pathogens tested did not rot green fruit.
Fruit inoculated with C. musae and held for 0, 1, 2 and 3 days at ambient temperature before ripening sustained increasingly more severe crown rot. C. musae was isolated with increasing frequency from rotting tissue the longer the period under ambient conditions. Crowns which had been inoculated with F. pallidoroseum and held for a comparable period did not display such extensive rot development although the pathogen was very frequently isolated from the tissue.     

Biocontrol of post-harvest fungal diseases on Dutch white cabbage by Pseudomonas and Serratia antagonists in storage trials

Post-harvest rotting of Dutch white cabbage was reduced by post-harvest treatment with Pseudomonas fluorescens strains CL42, CL66, CL82, Serratia plymuthica strain CL43 or Serratia liquefaciens CL80, in three storage trials carried out in an experimental cold store at Manchester University, Manchester, UK and in a commercial cold store at L.W. van Geest Farms Ltd, Spalding, UK. The amount of surface area covered by fungal growth was assessed at 6-week intervals during storage and the trimming losses were determined after 8 to 10 months. Only strains CL80 and CL82 were found to reduce fungal spoilage significantly in all three trials, and control by strain CL82 was similar to that achieved by post-harvest treatment with fungicides. In the commercial cold store, CL42 showed better results than any of the other bacterial strains.     

Antifungal activity, acid and sugar content in the wood apple (Limonia acidissima) and their relation to fungal development

Unripe wood apple fruit is generally free from visible fungal growth before and at harvest but a succession of fungi appears on the fruit shell, and sometimes in the pulp, during ripening. A TLC- Cladosporium bioassay of the chloroform extract taken from unripe fruit shell demonstrated three inhibition areas. Similar extracts from stem-bark and root-bark produced these three, and one additional, inhibition areas. The four compounds responsible for inhibition were identified as psoralene, xanthotoxin, 2,6-dimethoxybenzoquinone and osthenol.
Concentrations of the three antifungal compounds on unripe fruit shell increased during the first 4 days after harvest and then declined. They remained much below those required to inhibit the development of three fungi on TLC plates. Titratable acidity of the unripe fruit pulp was high but decreased by about 50% during ripening. Levels of reducing sugars were very low in the unripe fruit pulp but increased by about five times during ripening. Levels also increased in the fruit shell and its washings. The possible role of these factors in restricting fungal growth in unripe fruits is discussed.     

Early Events During Quiescent Infection Development by Colletotrichum gloeosporioides in Unripe Avocado Fruits

Inoculation of avocado pericarp tissue with Colletotrichum gloeospori-oides and treatment of avocado cell cultures with the cell wall elicitor of C. gloeosporioidesboth increased the production of reactive oxygen species (ROS). However, whereas the production of ROS could be detected within minutes in avocado cell suspensions, it was detected only after 2 h following inoculation of pericarp tissue. Protein kinase inhibitors such as K-252a and staurosporine and the phosphatase inhibitor microcystin-LR inhibited the release of H(2)O(2) from avocado cell suspensions. When 1 mM H(2)O(2) was exogenously applied to pericarp tissue, it enhanced ROS, phenyl-alanine ammonia lyase (PAL) activity, and epicatechin levels. But, when H(2)O(2) treatment was applied following staurosporine treatment, PAL activity was no longer induced. The uninduced ROS production in pericarp tissue of freshly harvested, unripe, resistant fruit was twice as high as in ripe, susceptible fruit. Challenge inoculation of resistant fruit further increased the ROS level; however, this increase did not occur in susceptible fruits. The current findings are consistent with the hypothesis that production of ROS is induced by fungal infection of unripe fruits and, consequently, may modulate resistance, resulting in the inhibition of fungal development and quiescence.     

The use of Trichoderma species to control strawberry fruit rots

The effect of temperature on the growth and antagonistic properties of Trichoderma species against Botrytis cinerea and Mucor mucedo (strawberry fruit pathogens) was studied. Five strongly antagonistic isolates were further used in field experiments. The incidence of pre-harvest rots caused by B. cinerea and the rate of post-harvest spoilage were similarly reduced when strawberry flowers were sprayed either with the fungicide dichlofluanid or with spores of selected Trichoderma isolates.     

Histological investigation of stripe rust (Puccinia striiformis f.sp. tritici) development in resistant and susceptible wheat cultivars

The wheat cultivar Kariega expresses complete adult plant resistance against stripe rust, whereas cv. Avocet S is susceptible. Using confocal laser scanning microscopy, initial fungal penetration into flag leaves was identical in both cultivars, with directional germ-tube growth towards stomata that were penetrated without the formation of an appressorium, followed by differentiation of a substomatal vesicle, infection hyphae, haustorial mother cells and haustoria. During the following 4 days, further fungal development occurred more quickly in the resistant than in the susceptible cultivar. However, by 7 days postinoculation (dpi) the situation changed, with exponential growth of the pathogen occurring only in the susceptible line. Induced cellular lignification, a typical defence reaction of cereals, was observed at 4 dpi in the resistant cultivar, and 2 days later lignified tissue completely surrounded the fungal colonies. In the susceptible cultivar, isolated lignified host cells occurred at 6 dpi, and long, unbranched fungal hyphae outgrowing the resistance reaction were observed.     

甜瓜果实表面生防芽孢杆菌的类群与鉴别

 从不同甜瓜果实表面可分离到在LB培养基上生长迅速、菌落形态呈明显差异的4类革兰氏染色阳性细菌。经过生理生化指标的鉴定、菌株间16S rDNA序列和部分特异性基因序列扩增以及种间遗传距离分析,将这4类芽孢杆菌分别鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和枯草芽孢杆菌(Bacillus subtilis)的3个形态变种。这些芽孢杆菌经继代培养20代后,仍能保持稳定的菌落形态和对灰葡萄孢(Botrytis cinerea)、链格孢(Alternaria alternata)、尖孢镰刀菌(Fusarium oxysporum)、黑曲霉(Aspegillus niger)和粉红单端孢(Trichothecium roseum)等8种果蔬采后病原真菌显著且广谱的拮抗作用。结果表明,甜瓜果实表面广泛分布着具有拮抗性能的枯草芽孢杆菌和其近缘种解淀粉芽孢杆菌,可通过特定培养条件下(LB培养基上37℃培养48h)单菌落的生长速度和菌落外观形态特征快速分离并鉴别它们的类群,为开发甜瓜果实采后保鲜制剂提供理论依据。     

Effect of crop growth and canopy filtration on the dynamics of plant disease epidemics spread by aerially dispersed spores

Most mathematical models of plant disease epidemics ignore the growth and phenology of the host crop. Unfortunately, reports of disease development are often not accompanied by a simultaneous and commensurate evaluation of crop development. However, the time scale for increases in the leaf area of field crops is comparable to the time scale of epidemics. This simultaneous development of host and pathogen has many ramifications on the resulting plant disease epidemic. First, there is a simple dilution effect resulting from the introduction of new healthy leaf area with time. Often, measurements of disease levels are made pro rata (per unit of host leaf area or total root length or mass). Thus, host growth will reduce the apparent infection rate. A second, related effect, has to do with the so-called "correction factor," which accounts for inoculum falling on already infected tissue. This factor accounts for multiple infection and is given by the fraction of the host tissue that is susceptible to disease. As an epidemic develops, less and less tissue is open to infection and the initial exponential growth slows. Crop growth delays the impact of this limiting effect and, therefore, tends to increase the rate of disease progress. A third and often neglected effect arises when an increase in the density of susceptible host tissue results in a corresponding increase in the basic reproduction ratio, R(0), defined as the ratio of the total number of daughter lesions produced to the number of original mother lesions. This occurs when the transport efficiency of inoculum from infected to susceptible host is strongly dependent on the spatial density of plant tissue. Thus, crop growth may have a major impact on the development of plant disease epidemics occurring during the vegetative phase of crop growth. The effects that these crop growth-related factors have on plant disease epidemics spread by airborne spores are evaluated using mathematical models and their importance is discussed. In particular, plant disease epidemics initiated by the introduction of inoculum during this stage of development are shown to be relatively insensitive to the time at which inoculum is introduced.     

Effect of light and dark on the growth and development of downy mildew pathogen Hyaloperonospora arabidopsidis

Disease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant–oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation, and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hr. In contrast, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development, and oospore formation, whereas exposure to constant light stimulated these three stages of development. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.