Evidence for Resistance-inducing Compounds in Conidia of Erysiphe graminis f.sp. hordei

Preparations of Erysiphe graminis f.sp. hordei conidia were spray-applied to the first leaf of barley plants which were subsequently challenge inoculated with virulent powdery mildew. The powdery mildew reducing effect of the preparations was assessed by scoring the outcome of the challenge inoculation. Homogenates of ungerminated conidia, germinated conidia, and methanol-water extracts of germinated conidia reduced the number of powdery mildew colonies. Cell wall fragments from ungerminated conidia, germinated conidia, and conidial germination fluid obtained from conidia germinated in aqueous suspension did not reduce the number of powdery mildew colonies. Microsconical analysis of the infection course following challenge inoculation indicated that the powdery mildew reducing effect is due partly to induced resistance.     

Conidia of the tomato powdery mildew <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> initiate germ tubes at a predetermined site

The emergence of germ tubes from the conidia of powdery mildew fungi is the first morphological event of the infection process, preceding appressoria formation, peg penetration and primary haustoria formation. Germination patterns of the conidia are specific in powdery mildew fungi and therefore considered useful for identification. In the present study, we examined conidial germination of the tomato powdery mildew Oidium neolycopersici KTP-01 in order to clarify whether germ tube emergence site in KTP-01 conidia is determined by the first contact of the conidia to leaves (as found for the conidia of barley powdery mildew), or alternatively is predetermined and is unrelated to contact stimulus. Highly germinative conidia of KTP-01 were collected from conidial pseudochains on conidiophores in colonies on tomato leaves using two methods involving an electrostatic spore attractor and a blower. In the electrostatic spore attraction method, the conidia were attracted to the electrified insulator probe of the spore collector—this being the first contact stimulus for the conidia. In addition, the blowing method was used as a model of natural infection; pseudochain conidia were transferred to detached leaves by air (1 m/s) from a blower. Thus, landing on the leaves was the first contact for the conidia. Furthermore, conidia were also blown onto an artificial membrane (Parafilm-coated glass slides forming a hydrophobic surface) or solidified agar plates in Petri dishes (hydrophilic surface). Eventually, almost all conidia on the probe and on tomato leaves or artificial hydrophobic and hydrophilic surfaces synchronously germinated within 6 h of incubation, indicating that the first contact of the conidia with any of the aforementioned substrata was an effective germination induction signal. Germ tube emergence sites were exclusively subterminal on the conidia. Moreover, the germ tubes emerged without any relation to the sites touched first on the conidia. Thus, the present study strongly indicates that conidia of O. neolycopersici produce germ tubes at a predetermined site.     

Powdery Mildew on Mulberry in Yunnan,China is Distinct from Other Phyllactinia spp. on Morus

The occurrence of an epidemic outbreak of a powdery mildew disease on mulberry in Yunnan province, China, is reported. Its symptoms are characteristic for powdery mildews and visible as white pathches covering the abaxial surfaces of leaves leading to chlorosis and necrosis. The pathogen is morphologically barely distinguishable from Phyllactinia moricola. However, it exhibits several new morphological characteristics which 2–3 conidia could be formed in short chains at the apex of the conidiophores and the conidia could produce two germ tubes in any position. Phylogenetic analyses of ITS sequences show that the pathogen has a close genetic relationship with P. moricola and Ph. broussonetiae‐kaempferi, two species on hosts belonging to family Moraceae. However, the ITS differences between Japanese sequences and the Chinese sequence derived from mulberry are greater than expected for a single species and suggest a cryptic species in China, but the present data are not sufficient for a final conclusion. Therefore, the Morus powdery mildew in Yunnan can currently only be classified as Phyllactinia sp. Morphological features, including conidial germination pattern of this powdery mildew are described in detail, and the local climatic conditions of the disease are analysed, which will provide the base for finding an effective method, including bio‐control, to control the disease under local conditions.     

An important role for secreted esterase in disease establishment of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici

The activity of esterase secreted by conidia of wheat powdery mildew fungus, Blumeria graminis f.?sp. tritici, was assayed using indoxyl acetate hydrolysis, which generates indigo blue crystals. Mature, ungerminated, and germinating conidia secrete esterase(s) on artificial media and on plant leaf surfaces. The activity of these esterases was inhibited by diisopropyl fluorophosphate, which is selective for serine esterases. When conidia were inoculated on wheat leaves pretreated with diisopropyl fluorophosphate, both appressorial germ tube differentiation and symptom development were significantly impaired, indicating an important role of secreted serine esterases in wheat powdery mildew disease establishment.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Germination of Erysiphe gratninis f.sp. hordei conidia on barley leaves

Germination of Erysiphe graminis f.sp. hordei conidia on leaves of several barley cultivars was studied in the laboratory. On both detached leaves and intact plants, within 48 h of inoculation a higher proportion of conidia had germinated on the basal and middle portions of the adaxial leaf surface than on the corresponding portions of the abaxial surface. Such differences between surfaces were not observed near the leaf tip. Similar results were obtained with all the cultivars and growth stages tested, and with five isolates of E. graminis, and are consistent with the observation that there is usually less powdery mildew on the abaxial than the adaxial surface of barley leaves. With most of the barley genotype/mildew isolate combinations tested, within 48 h of inoculation higher proportions of conidia germinated on seedlings and juvenile plants than on older plants. Inherited characteristics which affect spore germination on the leaf surface may be important factors in the development of adult-plant resistance of barley to powdery mildew, particularly in certain genotypes.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

Potential of Lecanicillium species for dual microbial control of aphids and the cucumber powdery mildew fungus, Sphaerotheca fuliginea

Detached leaf disc bioassays were conducted against cucumber powdery mildew and three species of aphid with three entomopathogenic species of Lecanicillium; Lecanicillium longisporum (Vertalec^®), Lecanicillium attenuatum (CS625), and an unidentified isolate (DAOM198499). The three Lecanicillium species had high virulence against the aphids Myzus persicae, Macrosiphum euphorbiae and Aulacorthum solani with the exception of DAOM 198499, which demonstrated reduced virulence to A. solani with an LT₅₀ of 6.4 days. Otherwise, LT₅₀ ranged between two and four days. Suspensions of conidia and blastospores of the Lecanicillium species were also applied onto 15 mm leaf discs dissected from cucumber plants previously inoculated with Sphaerotheca fuliginea. Powdery mildew did not develop when the Lecanicillium applications were made one and eight days after S. fuliginea inoculations. When Lecanicillium was applied to highly infected leaf discs 11 and 15 days after S. fuliginea inoculation, the application suppressed subsequent production of S. fuliginea spores as compared to the controls. These results suggest the potential of a dual role for Lecanicillium spp. as microbial control agents against aphids and powdery mildew.     

The effectivity of Tilletiopsis albescens in biocontrol of powdery mildew

Tilletiopsis albescens grows well on powdery mildew fungi inoculated on barley or cucumber leaves and causes collapse of the colonies. Application of ballistospores or cut mycelium was equally effective for biocontrol, and the effectiveness tended to increase exponentially with the concentration of germinating units (conidia and cut mycelium) applied. Seventy percent relative humidity or more is required for effective biocontrol. Two applications of T. albescens in the period from 3 days before to 3 days after inoculation with powdery mildew were more effective than one. Applications before inoculation or 7 days after inoculation with powdery mildew had little effect. T. albescens followed the powdery mildew as it was disseminated to uninoculated leaves, but this did not result in an effective biocontrol. The potential for using T. albescens for biocontrol of powdery mildews is discussed.     

A novel strain of Pseudozyma aphidis from mulberry parasitises the conidia of mulberry powdery mildew fungus Phyllactinia sp. and its biocontrol effect in the fields

Plant surface is colonised with a vast community of non-pathogenic epiphytic microorganisms which play an important role in host defence. In the present study, we reported a fungus from mulberry leaf surface that showed an antagonistic effect against mulberry powdery mildew fungal pathogen Phyllactinia sp. This novel isolate is a yeast-like fungus that was identified as Pseudozyma aphidis CNm2012 based on morphologic and phylogenetic analysis. According to our research, P. aphidis CNm2012 directly acted on the powdery mildew conidia via parasitism which caused conidial atrophy, collapse and eventually, cleavage and death. During the parasitic process, we found the isolate could gather around the conidia of Phyllactinia sp. and form hyphae that grew on the conidial surface and utilise the conidia as a nutrient source. Field studies revealed that P. aphidis CNm2012 could suppress the disease incidence of mulberry powdery mildew caused by Phyllactinia sp., and reduce the disease severity. To our knowledge, it is the first report of P. aphidis directly against powdery mildew fungus Phyllactinia spp. by parasitism. Our results indicated that P. aphidis CNm2012 could be served as an environmentally friendly alternative of chemical pesticides to manage mulberry powdery mildew disease.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

Inoculum production and long-term conservation methods for cucurbits and tomato powdery mildews

The behaviour of cucurbit powdery mildews (Podosphaera xanthii and Golovinomyces cichoracearum) and tomato powdery mildew (Oidium neolycopersici) infesting detached cotyledons of Lagenaria leucantha cv. ‘Minibottle’ was studied in order to develop an easy culture method for pure inoculum production. High spore production was found with a combination of mannitol (0.1 m), sucrose (0.02 m) and agar (8 g l⁻¹) in the cotyledon survival medium. Sporulation on cotyledons and viability of conidia were affected by the age of culture for the three species of powdery mildew tested. The age of cotyledons had also an impact of the spore production. This method was used to produce large amounts of inoculum for P. xanthii, G. cichoracearum and O. neolycopersici and enable the development of other species of powdery mildew like Leveillula taurica. Freezing conidia in liquid nitrogen enabled the long-term conservation of P. xanthii without any loss of virulence. The same method was unsuccessful with G. cichoracearum, and L. taurica and partly successful with O. neolycopersici.     

Leaf growth characteristics of Vitis vinifera and prediction of final lamina length for studies of powdery mildew

Total functional leaf area is a key factor in determining crop yield. A nonlinear mixed‐effects model was employed to estimate growth responses for individual leaves using repeated measures of lamina length ≥30 mm, in the absence of disease. Resulting growth curves make allowances for, and allow assessment of, leaf to leaf variability. The major source of variability in leaf growth was identified as differences in thermal time required to reach half final lamina length. Juvenile leaves of Vitis vinifera are susceptible to infection by the powdery mildew fungus (Erysiphe necator) which impairs leaf function. The model was used to predict unobserved final lamina length for a subset of leaves inoculated with E. necator immediately after observations ceased. The severity of infection by E. necator varies among infected leaves. A previous study identified which of the inoculated leaves developed symptoms of severe powdery mildew. Maximum severity of infection was found to occur when individual leaves were at 85.3–97.9% of predicted final lamina length.     

New records of microcyclic conidiogenesis in some powdery mildew fungi

Microcyclic conidiogenesis (MC) was recently described in several species of powdery mildew fungi. This process, defined as the production of conidia on a fungal spore without any, or only a minimal, involvement of hyphal growth, was observed on powdery mildew conidia that have already germinated on host plant surfaces and have been attached to the epidermal cells. Most probably, MC contributes to a quick propagation of young powdery mildew colonies because new conidia are sometimes produced in a shorter time on microcyclic conidiophores than on the hyphae of the young mycelium. This article reports MC in Erysiphe necator ex grapevine, Podosphaera leucotricha ex apple, Golovinomyces orontii ex tobacco, and Neoerysiphe galeopsidis ex Lamium purpureum based on light and low-temperature scanning electron microscopic studies.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

Mango powdery mildew Oidium mangiferae an alternative food for the predatory mites Typhlodromus mangiferus and Typhlodromips swirskii (Phytoseiidae) in absence or presence increasing prey density of Oligonichus mangiferus (Tetranychidae) in Egypt

The predacious mites, Typhlodromus mangiferus Zaher and El-Borolossy and Typhlodromips swirskii (Athias-Henriot), reproduced successfully on mango powdery mildew Oidium mangiferae Berthet in absence or presence of spider mite prey Oligonichus mangiferus (Rahman and Sapra) under laboratory conditions of 25 ± 1°C and 60–65% R.H. Adult female of both predators consumed protonymphs of O. mangiferus at different experimental densities. The consumption rate increased with increasing prey densities up to 25 protonymphs/female/day and decreased significantly at 35 and 50 protonymphs/female/day for the two predatory mites. Addition of powdery mildew conidia to each prey density significantly reduced consumption of spider mites at 35 and 50 protonymphs/female/day. Mean eggs/female/day by T. swirskii and T. mangiferus was 0.30 and 0.72 when reared on powdery mildew conidia compared with 1.64 and 1.57 when fed on powdery mildew and tetranychid prey, respectively. This increase in reproduction would have compensated the reduction in protonymph prey consumption due to the presence of mildew conidia. Mite–mildew interactions are discussed.     

Temporal Dynamics of Powdery Mildew (Oidium neolycopersici) and its Effects on the Host Growth Dynamics of Tomato

Controlled glasshouse experiments were conducted to investigate the temporal progress of powdery mildew and its effects on host dynamics of tomato, without and with one fungicide application. Healthy tomato transplants (5‐ to 6‐week old) were artificially inoculated with powdery mildew, and disease progress as well as host growth were monitored in both fungicide sprayed and unsprayed treatments and compared with non‐inoculated plants. Actual disease severity on a plant basis increased in unsprayed plants reaching maximum severity in the proportionate range of 0.53–0.83. One fungicide spray significantly reduced the maximum disease severity by two‐ to fourfolds. Despite adjustments for defoliation, declines in the proportion of disease severity between successive assessments were evident. Whereas the estimated growth rates of diseased plants were significantly lower than that of healthy plants, no significant differences were observed in the maximum leaf area formed of inoculated and non‐inoculated plants. A considerable effect of the powdery mildew epidemics was manifested through hastened shrivelling and defoliation of diseased leaves within the tomato canopy. An average of 18–29% and 40–52% of leaves had abscised from the plant canopy at the last date of assessment in sprayed and non‐sprayed plants, respectively. Accordingly, defoliation accounted for 14–33.3% and 58.3–63.1% losses in leaf area of sprayed and non‐sprayed plants, respectively. Duration of healthy leaf area and yield of inoculated plants were also significantly reduced by powdery mildew epidemics.     

Chronopathological Aspects of Disease Incidence in Rice (Oryza sativa L.)

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24 h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.     

The Infection Process of Pestalotiopsis Longisetula Leaf Spot on Strawberry Leaves

Pestalotia leaf spot, caused by the fungus Pestalotiopsis longisetula Guba, has become the major disease affecting strawberry production in Brazil. Strawberry seedlings with 4–5 leaves were inoculated with a conidial suspension of P. longisetula (2 × 10⁵ conidia/ml), and leaf samples were collected at 48, 72, 96 and 144 h after inoculation (hai) for observation in the scanning electron microscope. Conidia germinated within 48 hai. At 72 hai, conidia had formed very long germ tubes over the epidermal cells without any evidence of appressorial formation nor direct penetration. At 96 hai, fungal hyphae grew inter‐ and intracellularly in the lacunous parenchyma and also through tracheary elements. Pycnidia were first observed on the leaf surface at 96 hai. At 144 hai, conidia of P. longisetula were first liberated from the pycnidia. This study adds new information to better understand of the infection process of P. longisetula that may help in developing more effective disease control strategies.