Fruit Exocarp Phenols in Relation to Quiescence and Development of Monilinia fructicola Infections in Prunus spp.: A Role for Cellular Redox?

ABSTRACT Monilinia fructicola causes brown rot of Prunus species and usually remains quiescent on immature fruit but reactivates when fruit are mature. The dihydroxycinnamates caffeic acid and its quinate ester, chlorogenic acid, abundant in the exocarp of peach fruit, had no effect on fungal growth but markedly inhibited the production of the cell wall degrading enzymes polygalacturonase and cutinase in M. fructicola cultures. This inhibition was related to changes in the electrochemical redox potentials of the cultures, as measured with a redox electrode. Fungal culture filtrates had lower electrochemical redox potentials when the growth medium contained caffeic acid than in caffeic acid-free medium. Levels of total intracellular glutathione, the reduced form of which serves as a major cellular antioxidant, increased significantly in M. fructicola cells in response to external caffeic acid. The presence of caffeic acid, chlorogenic acid, or reduced glutathione in conidial suspensions of M. fructicola did not inhibit germination on flower petals and fruit, but inhibited appressorium formation from germinated conidia and subsequent brown rot lesion development. These results suggest that intracellular antioxidant levels in the pathogen can be influenced by phenols present in host tissue and that changes in the redox environment may influence gene expression and differentiation of structures associated with infection by the pathogen. The possible relationship of host phenols to quiescence and subsequent development of M. fructicola infections is discussed.     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development of Colletotrichum acutatum on Almond

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.     

Conidial germination, infection structure formation, and early colony development of powdery mildew on poinsettia

ABSTRACT Powdery mildew disease on poinsettias (Euphorbia pulcherrima) growing in commercial greenhouses was first observed in the United States in 1990 and has become an economically significant problem for poinsettia growers in the Midwest and northern United States since 1992. The temporal development of infection structures produced by conidial germ tubes of the pathogen (Oidium sp.) and the effect of high temperature on their development were investigated using poinsettia leaf disks placed in humidity chambers. Observations were made using light microscopy and scanning electron microscopy. At 20 degrees C (85% relative humidity), conidia germinated and formed an appressorium within 6 h of inoculation. Germination over time followed a monomolecular curve (r(2) = 0.77, P 相似文献   

Histological characterization of the early-stage infection events of Setosphaeria turcica in maize

Northern corn leaf blight (NCLB) caused by Setosphaeria turcica is a major foliar disease of maize. The early-stage infection events of this pathogen on maize leaves are unclear. We investigated the optimum temperature for conidial germination and appressorium formation, and characterized penetration and growth of S. turcica in maize leaf sheath and onion epidermis cells, including use of histological staining to assess plant cell viability. The results showed that the optimum temperature for conidial germination and appressorium formation was 20°C. On the maize leaf sheath, the appressoria were formed by germinated conidia, and penetration on the epidermal cells occurred at 8 h postinoculation (hpi). Round vesicles developed beneath the appressoria. Between 16 and 24 hpi, the branched invasive hyphae invaded three to five adjacent cells at most infection sites. The invasive hyphae tended to move along the cell wall and crossed from one cell to another. In the onion epidermis cells, the appressoria formed at 8 hpi, and in most cases the epidermal cells were penetrated through the juncture of the cell walls. At 16–24 hpi, the primary hyphal terminus swelled to a vesicle. The maize leaf sheath cells died at 8 hpi, whereas the onion cells did not. Our findings documented in detail the penetration and invasive hyphal growth in maize leaf sheath and onion epidermis, as well as viability of plant cells, at the early stages of infection, and provide a foundation for elucidating the underlying mechanism of S. turcica–maize interactions.     

Effect of humidity and temperature on conidial germination and appressorium development of two Philippine isolates of the mango anthracnose pathogen Colletotrichum gloeosporioides

A comparison of rates of germination and appressorium formation by an isolate of Colletotrichum gloeosporioides on mango leaves, fruit surfaces and cellophane membranes showed that behaviour was broadly similar on all three substrates. Frequency of appressorium formation was slightly higher on cellophane membranes, and both hyaline and melanized appressoria were formed. Only melanized appressoria were formed on mango surfaces. In vitro experiments on membranes showed comparative differences in physiological behaviour with temperature for two Philippine isolates of C. gloeosporioides . The most stimulatory temperature for production of appressoria differed in isolates I-2 and I-4 (25 and 20°C, respectively). At 30°C more appressoria became melanized than at lower temperatures, but the frequency of formation of penetration pegs was highest at 25°C. Conidia of C. gloeosporioides germinated on cellophane membranes at relative humidities as low as 95%, but the percentage of conidia germinating and forming appressoria increased as the RH approached 100%. Approximately 18% of conidia of C. gloeosporioides I-2 held at 62 and 86% RH for 4 weeks retained viability, and some were capable of forming appressoria when placed at 100% RH. These results have implications for epidemiological models for disease control.     

Light and Scanning Electron Microscopy Studies on the Infection of Oriental Lily Leaves By Botrytis Elliptica

Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer). At 20 °C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (< 20 m) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one-third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approximately 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica occurs via short swollen germ tube appressoria mostly near stomata.     

Calcium/calmodulin-dependent signaling for prepenetration development in <Emphasis Type="Italic">Cochliobolus miyabeanus</Emphasis> infecting rice

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca²⁺ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.     

Germination and Sporulation of Colletotrichum acutatum on Symptomless Strawberry Leaves

ABSTRACT The germination and sporulation of Colletotrichum acutatum were characterized over time on strawberry leaves (cv. Tristar) and plastic coverslips incubated at 26 degrees C under continuous wetness. Conidia germinated within 3 h after inoculation and formed melanized appressoria with pores by 9 h after inoculation. Host penetration was not observed up to 7 days after inoculation. Production of secondary conidia on conidial and hyphal phialides began within 6 h after inoculation. Secondary conidiation was responsible for up to a threefold increase in the total number of conidia within 7 days after inoculation. Primary conidia and hyphae began to collapse 48 h after inoculation, whereas melanized appressoria remained intact. These findings suggest that appressoria and secondary conidia of C. acutatum produced on symptomless strawberry foliage may be significant sources of inoculum for fruit infections.     

Inoculum dynamics, fruit infection, and development of brown rot in prune orchards in california

ABSTRACT Brown rot, caused by Monilinia fructicola, is a destructive disease of stone fruit in California. Disease management requires information on inoculum dynamics and development of latent and visible fruit infections during the season to help make decisions on timing of fungicide treatments and choice of cultural practices. In this study, the daily spore concentration (ascospores and conidia) of M. fructicola in the air was monitored with spore traps in two prune orchards during the growing seasons in 2001 and 2002. The spore concentrations were low to moderate at early bloom, increased at full bloom, and decreased to the lowest level at the end of bloom. Improper timing of fruit thinning and irrigation in midseason increased spore concentration in the air and fruit infections late in the season. Artificial fruit inoculations were conducted periodically in 10 prune orchards in 2002 and 2004, and incidence of fruit rot at different inoculation dates was assessed. Fruit rot development rate increased linearly with inoculation date during the growing season. Natural blossom and fruit infections were monitored periodically in 10 prune orchards, and incidence of latent fruit infection was determined by using the overnight freezing-incubation technique. Incidence of fruit rot also was assessed 2 weeks before harvest in these orchards. The incidence of latent fruit infection at the pit hardening stage significantly correlated with that at the late stages and with the incidence of fruit rot at harvest.     

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.     

Early brown rot infections in sweet cherry fruit are detected by monilinia-specific DNA primers

ABSTRACT Visible and nonvisible quiescent infections of immature and mature fruit are an integral component of the disease cycle of brown rot of sweet cherry in California. Detection of these infections is critical for developing efficient and efficacious fungicide management programs. The previously published DNA amplification primers mfs3 and NS5 for the identification of Monilinia fructicola were very specific in amplifying DNA of M. fructicola only and not M. laxa. This primer set, however, only detected DNA from some of the California isolates of M. fructicola. This genetic diversity was supported by random amplified polymorphic DNA (RAPD) analysis. Using eight 10-mer primers, seven M. fructicola isolates from California were all identified as genetically distinct. Using the same primers, only one polymorphism was detected among seven isolates of M. laxa. The multiple genotypes identified within the small population sample of M. fructicola, but not of M. laxa, using RAPD analysis could be indicative of genetic recombination within M. fructicola but not within M. laxa. To detect early brown rot infections in fruit, two primer sets that were developed from DNA sequences of either ribosomal DNA (MF5/ITS4/ITS3) or a RAPD fragment (X-09intF3/X-09R) specifically amplified DNA from isolates of M. fructicola and Monilinia species, respectively. No amplification products were present when using DNA from Botrytis cinerea or from other fungi commonly found on sweet cherry fruit. Primers X-09intF3 and X-09R were more sensitive and reliable for detecting small amounts of target DNA either extracted from conidia or from laboratory-inoculated cherry fruit with early brown rot infections that showed no visual symptoms or with visible quiescent infections. Furthermore, these primers also were effective for detecting visible quiescent infections in cherry fruit that were collected in the field.     

The relationship between antifungal diene levels and fungal inhibition during quiescent infection of unripe avocado fruits by Colletotrichum gloeosporioides

Quantitative changes in the antifungal compound, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12.15-diene, in freshly harvested avocado fruits during the initial stages of fungal development were investigated to determine the possible involvement of the compound in quiescent infections of Colletotrichum gloeosporioides. The concentration of the antifungal compound in the peel decreased to subfungitoxic concentrations 16 h after harvest. Fifty-six hours later the antifungal diene had increased to c . 3800 μg/g fresh weight. At this stage, germinated appressoria had penetrated the cuticle to the epidermal cells but no fungal development was observed until 7 days later when the concentration of the diene had decreased to 100-110μg/g fresh weight. Following a dip treatment at 55°C for 5 or 10 min, the antifungal diene concentration decreased as in the controls, but it remained at subfungitoxic concentrations for a longer period enabling fungal development and early symptom expression.
The concentration of the diene in the flesh of freshly harvested fruit decreased to 120 μg/g fresh weight 24 h after harvest. Inoculation of peeled fruits with spores of C. gloeosporioides showed germination without appressoria formation and symptom expression occurred 24-48 h later. Symptom expression was delayed if fruits were inoculated after coating the flesh with epicuticular wax extracts or if the flesh was inoculated 3 days after harvest when the antifungal diene had regained a fungitoxic concentration. Disease symptoms were expressed in soft fruits containing subfungitoxic concentrations of the diene.
We conclude that the diene in unripe avocado fruits inhibits fungal development of germinated appressoria or conidia. The quiescent structure of C. gloeosporioides in unripe avocado fruit is a subcuticular hypha.     

Cytological events in tomato leaves inoculated with conidia of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f. sp. <Emphasis Type="Italic">hordei</Emphasis> and <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.     

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.     

小麦的种和品种对白粉病抗性的初步研究

 在对白粉病具不同抗性的11个小麦的种和品种上进行的按种试验表明,在接种后8小时内,分生孢子达到最高萌发率(约为60%),并形成附着孢。在抗病的和感病的小麦叶片上,分生孢子的萌发率和萌发速度没有差异。接种后12小时,在感病的材料上,发现真菌侵入寄主,但在抗病的材料上,发现最早侵入时间,至少要比在感病材料上约晚4小时。接种后36小时,在高感的材料上,萌发的分生孢子有83%已侵入寄主,其中约有70~80%已形成吸胞。在感病、抗病和高抗材料上的侵入率,分别为70,45,27%。形成或开始形成吸胞的分别为55~61,14~27,4~8%。在抗病材料的表皮细胞内,真菌形成的吸胞较小,并有点畸形。在抗病材料的叶面上,真菌的菌丛稀薄,产孢也很少。     

Secondary inoculum dynamics of Monilinia spp. and relationship to the incidence of postharvest brown rot in peaches and the weather conditions during the growing season

Eight field surveys over three growing seasons (April to October in 2006, 2007, and 2008) were done in three commercial peach and nectarine orchards in order to determine the secondary inoculum dynamics of Monilinia spp. and relationship to the weather conditions and incidence of postharvest brown rot in the Ebro Valley, which is the main peach fruit growing region in Spain. After regression analysis of the epidemiological data of postharvest brown rot and the climatic variables, a disease outbreak can be predicted from (i) the times of the first appearance of airborne conidia, the first appearance of conidia on the surface of flowers and fruits, and the first latent infection, all of which occur 2?months before harvest (ii) the number of conidia on the fruit surface, 2?weeks and 1?week before harvest, (iii) the preharvest incidence of brown rot, and (iv) the mean environmental temperature from popcorn to harvest. From these results, we confirmed the importance of the secondary inoculum dynamics of Monilinia spp. and the utility of these dynamics to predict an outbreak of brown rot in peaches on the day of their harvest and after their harvesting.     

Conidial density of <Emphasis Type="Italic">Monilinia</Emphasis> spp. on peach fruit surfaces in relation to the incidences of latent infections and brown rot

To evaluate the effect of conidial density of Monilinia spp. on the fruit surface on the incidence of latent infection and brown rot in peaches, eleven field surveys were performed in commercial orchards located in Cataluña, Spain over four growing seasons from 2002 to 2005, and nine surveys were conducted to determine the sources of overwintered Monilinia spp. inoculum. There was a significant positive relationship (r?=?0.69) between the numbers of conidia of Monilinia spp. on the fruit surface and the incidence of latent infections, but not with brown rot at harvest. Although mummified fruit, twigs and pits have been identified as being able to carry the pathogen from year to year in peaches grown in Spanish orchards, no relationships between any of these sources and the numbers of conidia on the fruit surface, or incidence of latent infection or brown rot were found. The effect of temperature (T), solar radiation (SR), rainfall (R) and wind speed (WS) on the area under the number of conidia of Monilinia spp. curve (AUncC) on peach surfaces was analysed. Regression analysis revealed that T, SR, R, and WS could account for 99% of the total variation in the area of the AUncC on peach surfaces. Thus, in order to reduce the incidence of latent infection and brown rot it is essential not only to remove the sources of primary inoculum but also to reduce the number of Monilinia spp. conidia on the fruit surface. Furthermore, the sources of airborne conidia of Monilinia spp. should be taken into consideration in disease management programmes in Spain.