Production Of Pycnidia Research Articles

Diaporthe foeniculina and D. eres, in addition to D. ampelina, may cause Phomopsis cane and leaf spot disease in grapevine.

Phomopsis cane and leaf spot (PCLS) disease, affecting grapevines (Vitis vinifera and Vitis spp.), has been historically associated with Diaporthe ampelina. Typical disease symptoms, comprising bleaching and black pycnidia, have also been associated with other Diaporthe spp. In this study, we conducted a molecular identification of the Diaporthe isolates isolated from grapevine canes from different geographic areas of southern Europe showing PCLS symptoms. Then, we investigated their morphological characteristics (including mycelium growth and production of pycnidia and alpha and beta conidia) in response to temperature. Finally, we artificially inoculated grapevine shoots and leaves with a subset of these isolates. Based on our results, PCLS etiology should be reconsidered. Though D. ampelina was the most crucial causal agent of PCLS, D. eres and D. foeniculina were also pathogenic when inoculated on green shoots and leaves of grapevines. However, D. rudis was not pathogenic. Compared to D. ampelina, D. eres and D. foeniculina produced both pycnidia and alpha conidia at lower temperatures. Thus, the range of environmental conditions favorable for PCLS development needs to be widened. Our findings warrant further validation by future studies aimed at ascertaining whether the differences in temperature requirements among species are also valid for conidia-mediated infection since it could have substantial practical implications in PCLS management.

Temperature-Dependent Sporulation of the Fungus Coniella diplodiella, the Causal Agent of Grape White Rot.

White rot, caused by the fungus Coniella diplodiella, can severely reduce grapevine yields worldwide. Currently, white rot control mainly relies on fungicides applied on a calendar basis or following hailstorms that favor disease outbreaks; however, the control achieved with this strategy is often inconsistent or otherwise unsatisfactory. Realizing more rational control requires an improved understanding of white rot epidemiology. To this end, we conducted experiments with grapevine berries of two Vitis vinifera cultivars (either injured or not before artificial inoculation with a conidial suspension of C. diplodiella) to determine the effect of temperature on the length of latency (i.e., the time between infection and onset of mature pycnidia on berries) and the production of pycnidia and conidia. Sporulation occurred between 10 and 35°C, with the optimum detected at 20°C. The latency period (LP) was shorter at 25 to 35°C than at lower temperatures; the shortest LP was 120 h at 30°C on injured berries. Affected berries produced abundant conidia at 15 to 30°C (the optimum was 20°C) for more than 2 months following inoculation. Mathematical equations were developed that fit the data, with strong associations with temperature for the LP (R2 = 0.831) and for the production dynamics of secondary conidia (R2 = 0.918). These equations may contribute to the development of a risk algorithm to predict infection periods, which can inform risk-based disease control strategies rather than calendar-based disease control strategies.

First Report of Cytospora ailanthicola Causing Canker on cherry blossom (Cerasus serrulata) in China.

Cherry blossom (Cerasus serrulata) is a plant with important garden applications. It is a newly introduced exotic plants in the Arar region of Xinjiang, China (40°41'18.19″N,81°43'50.55″E). In October 2022, it was discovered that about 30% of cherry blossoms had a canker disease. The leaves of the sick branches were dired, the branches themselves were damaged, with dark brown color inside. Orange-yellow conidia horns were produced in humid condition. Samples were collected from fifteen trees exhibiting notable symptoms. The diseased junctions of the infected shoots were chopped into small pieces and subjected to surface sterilization by using 70% ethanol for 30s, 1% NaClO solution for one minute, and sterile distilled water three times (Chen et al. 2016). The representative strain YINGHUA-1 was chosen for identification by molecular biology and morphology. After five days of incubation at 26℃ on PDA media, colonies of white fluffy mycelium were produced from the YINGHUA-1 strain. After 25 days of PDA culture, the production of pycnidia was first observed, circular, black. The pycnidia began to produce conidia at 30 days. The conidia was translucent without septum, with a slightly curved single cell and smooth surface. Pycnidia was spherical and flat, with a single black aperture at the top that resembles a nearly round hole, the chamber was made up of several tiny chambers separated by a shared wall, and its diameter ranges from 900-1900 µm. The size of the conidium was 3.7-6.6×1.1-1.9 μm (n=20). The intrinsic transcriptional spacer (ITS), transcriptional elongation factor (tef-1α), and β-tubulin (tub2) gene moieties of rDNA were sequenced using ITS1/ITS4, EF1-728F/EF1-986R, and Bt2a/Bt2b primers, respectively(Zhang et al. 2014). The amplified sequences of ITS region (Accession No. OR855907), tub2 (Accession No. OR865863) and tef-1α (Accession No. OR865864) were deposited in the GenBank. BLAST searches of the sequences revealed 99.59% identity (474/476 bp) of the ITS sequence, 98.63% identity (216/219 bp) of the tef-1α sequence, and 98.55% identity of the tub2 sequence (339/344 bp) with C. ailanthicola CFCC59446 (accessions OR826163, OR832040, and OR832062, respectively.) Phylogenetic analyses were performed with Iqtree v.1.6.12 for maximum likelihood (ML). Confidence levels for the nodes were determined using 1000 replicates of bootstrapping methods. Based on phylogenetic analysis and morphological characteristics, the pathogen was identified as C. ailanthicola. The pathogenicity of C. ailanthicola was confirmed by inoculation of 1-year-old shoots (5 replicates of this experiment). After 7 days, symptoms of inner bark discoloration were visible on xylem of branches and the same fungus was re-isolated from the inoculated shoots, with no lesions on the control shoots. C. ailanthicola is only known from a single host plant, Ailanthus altissima,in China (Fan et al.2020). As far as we know, this is the first report of C. ailanthicola harmings C. serrulata in China.

First report of Leptosphaeria sclerotioides on Brassica napus in Northwestern China

In 2020, diseased seedlings of winter oilseed rape (Brassica napus) with cankered taproots as well as abundant sclerotium-like structures in the soil surrounding the roots were found in Longxi County of Gansu province of northwestern China. A fungus with production of pycnidia was isolated from the diseased roots, and it was identified based on morphological characteristics, molecular phylogeny (ITS, LSU) and PCR detection with the specific primers. The fungus was identified as Leptosphaeria sclerotioides Gruyter, Aveskamp & Verkley [anamorph: Phoma sclerotioides (Preuss) ex Sacc.]. Re-inoculation of isolates of P9 and P10 of L. sclerotioides on winter oilseed rape (B. napus cultivar ‘Zhongshuang No. 9’) in Wuhan caused formation of abundant sclerotium-like structures in soil surrounding the roots, but failed to produce root cankers as those observed in Gansu possibly due to lack of long peroid of low-temperature conditioning in Wuhan. In spite of this, plant height, pod number and seed yield of oilseed rape were significantly reduced in the treatment with L. sclerotioides P9 and P10, compared to the control treatment. To the best of our knowledge, this is the first report of L. sclerotioides on B. napus in China and the finding broadened our understanding about the natural distribution of this psychrophilic fungus.

First report of Diplodia mutila causing wood necrosis in European hazelnut in Chile.

In Chile, the planted area of European hazelnut (Corylus avellana L.) reaches around 30,000 hectares, mainly concentrated in the central and southern area of the country where climate and soil provide a natural environment well suited to growing this species. Only a few diseases affect this nut tree in Chile. During the spring seasons in 2018 and 2020, European hazelnut plants (6 to 20% of incidence) exhibited wood necrosis and vascular discoloration of branches, with reduced growth, cankers and wilt branches, in orchards located in San Clemente and Curicó, Maule Region, Bulnes and El Carmen, Ñuble Region, Chile (36°45'-36°54' S; 71°03'-72°26' W). Symptomatic tissues were surface disinfected using a ~1% commercial sodium hypochlorite solution. Disinfected tissues were cut longitudinally, placed onto potato dextrose agar (PDA, Difco) plates, and incubated at 25 °C in the dark for 48 hours. Fungal hyphal tips were taken and placed on PDA medium. A fungal species was consistently isolated from these lignified tissues. The mycelium was initially translucent (turning white in appearance), while the mature mycelium was aerial, varying in color from pale to dark gray (Munsell color code: colony edge mycelium 6Y-6 4 / 5G and colony center mycelium B6-PB 7 / 5PB). The production of pycnidia and conidia was induced using pine needles in water agar medium and incubated in the dark for 10 days. Hyaline unicellular conidia of 25 ± 1.1 µm (range 23.9 to 26.1 µm) long and 11 ± 0.5 µm (Range 10.5 to 11.5 µm) wide (n = 50) were obtained from black pycnidia. Based on the cultural and morphological characteristics observed, the pathogen was identified as a possible species of the family Botryosphaeriaceae (20 isolates). Molecular techniques were used to identify the species of pathogen, and three isolates (F154, F199, and F167) were analyzed by using Multilocus sequence typing to confirm the identity of the pathogen. Genes ITS (internal transcribed spacer region), tef-1 (translation elongation factor 1-alpha) and β-tub (β-tubulin) were amplified using endpoint PCR, with primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Carbone & Kohn, 1999) and Bt2a/Bt2b (Glass & Donaldson, 1995), respectively. The segments were sequenced using the same primers, deposited in Gen Bank, and the accession numbers for each isolate were OM993582, OM993583, ON003481 for ITS, ON054936, ON054938, ON054937 for tef1 and ON054939, ON054941, ON054940 for β-tub, respectively. A phylogenetic tree was constructed using the maximum likelihood statistical method with the Tamura-Nei model based on a concatenated dataset of ITS region, tef1 and β-tubulin gene using Mega-X, and the three Chilean isolates (F154, F199, and F167) formed a single clade with the reference isolates of Diplodia mutila (Fr.) Mont. BLAST algorithm analyses indicated 100% identity to D. mutila for ITS (accession NR_144906), for tef-1 (accession MK573559), and for β-tubulin (accession MG952719). The pathogenicity of the three isolates was validated through Koch's postulates. For this purpose, a trial was established in 6-year-old European hazelnut plants cv. Tonda Di Giffoni. Ten healthy branches were individually inoculated using actively growing mycelial discs from each isolate, while a disc of PDA without fungus was used as a control. Holes of 5-mm diameter were inoculated, making sure the mycelium was in contact with the wood. Finally, the wounds were sealed with plastic film to prevent external contamination and improve humidity conditions. After 120 days, each branch was cut longitudinal-sectioned to verify the presence of wood necrosis which arose between 3.0 to 16.2 mm of length around the point of inoculation. No necrosis was observed in the control. To confirm pathogenicity, infected tissues were cut into small pieces with sterile knives and scalpels, and surface disinfected with a 1% sodium hypochlorite solution for 1 min. The disinfected tissues were placed on PDA medium and incubated at 25°C in the dark until fungal growth was observed. Hyphal tips were taken from the mycelia developed from the pieces of wood, and placed on PDA medium in order to obtain pure isolates. The pathogenicity of the D. mutila isolates F154 and F199 was observed in 100% of the inoculated branches, while isolate F167 showed symptoms in 85% of the branches. The reisolated strains showed similar mycelial growth and microscopic fungal structures to those observed in the isolates used for inoculation. This is the first report of D. mutila affecting European hazelnut in Chile. This fungus has been recently reported affecting hazelnut in Oregon, USA (Wiman et al., 2019), causing similar symptoms to those observed in our study. In addition, D. mutila has been reported infecting walnut in Chile (Diaz et al. 2018) and native forest trees, specifically Araucaria araucana in Chile (Besoain et al., 2017). The presence of D. mutila in commercial hazelnut orchards in Chile highlights the need for epidemiological studies in order to understand the characteristics and impact of this pathogen and, based on this, develop adequate phytosanitary programs for its control.

Biological control of cabbage head rot (Sclerotinia sclerotiorum) by Coniothyrium minitans

BackgroundSclerotinia sclerotiorum is the causal agent of white mould, stem and head rot diseases on a wide variety, including cabbage in the field and storage. Control of this pathogen by using commercial disease management methods is extremely difficult. Therefore, this study was performed to identify the potential biological control agent.ResultsThe antagonist fungal isolates, a mycoparasite of S. sclerotiorum, were isolated and described as KET Cm1, PAL Cm 2 and TDK Cm 3. They were subjected to morphological and microscopic observation. The growth of antagonist fungal isolates on different media revealed that PDA and half-strength Czapek dox agar medium showed the maximum mycelial growth (28.67 and 28.33 mm) and pycnidial production in the PDA medium. The studies on pH and temperature found that growth and sporulation of the antagonist fungal isolates were optimum at pH 5.5 (34 mm) and 20 °C. The molecular confirmation of antagonist fungal isolates was carried out using ITS 1 and ITS 4 primers, which target 18S-28S rRNA gene fragment. The isolates were partially sequenced and showed that more than 99% identity with Coniothyrium minitans already exist in the NCBI database. The accession numbers (MW093134, MW111282 and MW136938) were obtained for all the three isolates and also confirmed using specific primers Cm sp 1F and Cm sp 1R. Among the tested isolates, the antagonistic activity of C. minitans TDK Cm 3 showed the maximum mycelial growth inhibition of 48.8% S. sclerotiorum and was used for further studies. Penetration and formation of appressoria on the hyphae of S. sclerotiorum by C. minitans isolate TDK Cm 3 were observed through the scanning electron microscope. The ethyl acetate fractions of TDK CM 3 isolate were evaluated against S. sclerotiorum by agar well diffusion assay, paper disc assay and poison food technique in vitro. Through GC–MS analysis, the effective antifungal compounds were identified.ConclusionThe present study focused on the identification and characterization of effective Coniothyrium isolates as eco-friendly, integrated disease management strategies against cabbage head rot disease.

Endophytes of Brazilian Medicinal Plants With Activity Against Phytopathogens.

Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Resistance induction and biological control have been exploited in agriculture due to their enormous potential. In this study, we investigated the antimicrobial potential of endophytic fungi of leaves and petioles of medicinal plants Vochysia divergens and Stryphnodendron adstringens located in two regions of high diversity in Brazil, Pantanal, and Cerrado, respectively. We recovered 1,304 fungal isolates and based on the characteristics of the culture, were assigned to 159 phenotypes. One isolate was selected as representative of each phenotype and studied for antimicrobial activity against phytopathogens. Isolates with better biological activities were identified based on DNA sequences and phylogenetic analyzes. Among the 159 representative isolates, extracts from 12 endophytes that inhibited the mycelial growth (IG) of Colletotrichum abscissum (≥40%) were selected to expand the antimicrobial analysis. The minimum inhibitory concentrations (MIC) of the extracts were determined against citrus pathogens, C. abscissum, Phyllosticta citricarpa and Xanthomonas citri subsp. citri and the maize pathogen Fusarium graminearum. The highest activity against C. abscissum were from extracts of Pseudofusicoccum stromaticum CMRP4328 (IG: 83% and MIC: 40 μg/mL) and Diaporthe vochysiae CMRP4322 (IG: 75% and MIC: 1 μg/mL), both extracts also inhibited the development of post-bloom fruit drop symptoms in citrus flowers. The extracts were promising in inhibiting the mycelial growth of P. citricarpa and reducing the production of pycnidia in citrus leaves. Among the isolates that showed activity, the genus Diaporthe was the most common, including the new species D. cerradensis described in this study. In addition, high performance liquid chromatography, UV detection, and mass spectrometry and thin layer chromatography analyzes of extracts produced by endophytes that showed high activity, indicated D. vochysiae CMRP4322 and P. stromaticum CMRP4328 as promising strains that produce new bioactive natural products. We report here the capacity of endophytic fungi of medicinal plants to produce secondary metabolites with biological activities against phytopathogenic fungi and bacteria. The description of the new species D. cerradensis, reinforces the ability of medicinal plants found in Brazil to host a diverse group of fungi with biotechnological potential.

Comparison of different inoculation methods in the investigation of fungal trunk diseases in grapevines

Amaç: Asma gövde hastalıkları, birçok fungal bitki patojeninin neden olduğu kompleks hastalıklardır. Bu çalışmada dört farklı hastalığı (Petri ve Siyah Bacak Hastalığı, Botryosphaeria ve Phomopsis Geriye Ölüm Hastalıkları) temsil eden bazı fungal patojenler için en uygun inokulasyon yönteminin saptanması amaçlanmıştır.Yöntem ve Bulgular: Phaeomoniella chlamydospora, Dactylonectria macrodidyma, Neofusicoccum parvum ve Diaporthe ampelina türü funguslar dormant asma kalemlerine veya yeşil sürgünlere farklı yöntemlerle inokule edilmişlerdir. Dormant kalemlere uygulanan yöntemler; a) kabuk altına miselyal agar disk inokulasyonu, b) tepeden agar disk inokulasyonu, c) ksileme enjeksiyon yöntemi ve d) yeşil sürgünlere agar disk inokulasyonudur. Patojenlerin kalemler ve yeşil sürgünlere inokulasyonunu takiben 2 ay süreyle gelişmelerine izin verilmiş ve sonra dokularda oluşan lezyon uzunlukları ölçülmüştür. Sonuçlara göre tüm patojenler tüm inokulasyon yöntemleriyle, hem dormant kalemler hem de yeşil sürgünler üzerinde lezyonlar meydana getirmiş ancak ölçülen lezyon uzunlukları kullanılan yöntemlere göre farklılık göstermiştir. En uzun lezyonların oluştuğu patojen ve inokulasyon metodu eşleşmesi şu şekilde gerçekleşmiştir; Diaporthe ampelina için yeşil sürgüne miseliyal agar disk inokulasyonu (72.2 mm), D. macrodidyma ve P. chlamydospora için ksileme enjeksiyon (sırasıyla 23.0 ve 66.1 mm) ve Neofusicoccum parvum için kabul altına miseliyal agar disk inokulasyonu (51.5 mm).Genel Yorum: Asmalarda fungal gövde hastalıklarının araştırılmasında birçok araştırmacı tarafından farklı inokulasyon yöntemleri kullanılsa da, patojenisite testlerinin bu çalışmada denenen yöntemlerden herhangi biri ile yapılabileceği ortaya koyulmuştur. Çok sayıda spor üreten türlerle yapılan çalışmalarda, ksileme enjeksiyon yönteminin daha makul sonuçlar vereceği görülmüştür. Miselyal gelişimi güçlü ve piknit üreten türler için ise kabuk altına ya da tepeden agar disk inokulasyon yöntemlerinin daha uygun olduğu anlaşılmıştır.Çalışmanın Önemi ve Etkisi: Bir araştırmada, inokulasyon yönteminin seçimi o araştırmanın sonuçlarını doğrudan etkilemektedir. Bu çalışmanın sonuçlarının, asma gövde hastalıkları ile yapılacak araştırmalarda uygun bir inokulasyon yönteminin seçimine yardımcı olabileceği düşünülmektedir.

Oxalic Acid Metabolism Contributes to Full Virulence and Pycnidial Development in the Poplar Canker Fungus Cytospora chrysosperma.

Poplar Cytospora canker, which is mainly caused by Cytospora chrysosperma, is one of the most destructive and widespread tree diseases worldwide. Although oxalic acid (OA) is demonstrated as an important virulence determinant in several necrotrophic fungi, specific functions of OA during pathogenesis remain controversial. Here, we identified three genes (CcOah, CcOdc1, and CcOdc2) directly involved in OA biosynthesis and catabolism in C. chrysosperma. We demonstrated that CcOah is required for OA biogenesis. All three genes were found to be highly upregulated during early infection stages of the poplar stem. The deletion of any of the three genes led to an obvious reduction of pycnidial production but no abnormality of hyphal growth and morphology. Furthermore, the individual deletion strain exhibited significantly limited lesion sizes on poplar twigs and leaves. Exogenous application of OA or citric acid can complement the virulence defects of ΔCcOah and ΔCcOdc1 strains. We further found that the ΔCcOah strain strongly promoted reactive oxygen species burst of poplar leaves during infection. Finally, induced secretion of OA was observed by monitoring color change of the plates after poplar stem extracts were added in the cultures; however, we failed to quantify OA concentration by high-performance liquid chromatography. Taken together, the present results provide insights into the function of OA acting as an important virulence factor of C. chrysosperma.

A Simple and Effective Technique for Production of Pycnidia and Pycnidiospores by Macrophomina phaseolina.

Production of pycnidia and pycnidiospores by Macrophomina phaseolina is not often seen in vitro. The objective of this study is to develop a simple and effective technique to obtain pycnidiospores of M. phaseolina isolates in vitro and to evaluate germination rates and pathogenicity of pycnidiospores. We found M. phaseolina isolates can produce pycnidia on oatmeal agar (OMA) under ultraviolet light with 365 nm wavelength (UV). For evaluating the effect of OMA and UV on growth of M. phaseolina, combinations of two agar media and three lighting conditions were tested. The results confirm that all six M. phaseolina isolates produced pycnidia only on OMA under UV. The pycnidiospores produced on OMA under UV had germination rates higher than 90%. In pathogenicity tests, inoculation with the pycnidiospores showed symptoms later than inoculation with hypha-colonized toothpicks. Significant differences in the pathogenicity is detected between isolates Mp2014003 and Mp2014024 when inoculation is done with the pycnidiospores (P < 0.001), but not when hypha-colonized toothpicks are used as inoculum (P = 0.091). This study provides a new method for obtaining pycnidiospores of M. phaseolina for future investigations.

Ray blight of pyrethrum in Australia: a review of the current status and future opportunities

Ray blight caused by Stagonosporopsis tanaceti is one of the most important diseases of pyrethrum (Tanacetum cinerariifolium), a perennial herbaceous plant cultivated for the extraction of insecticidal pyrethrins in Australia. The disease is responsible for complete yield loss in severe outbreaks. Infected seed is considered as the principal source of S. tanaceti. Infection hyphae remain only in the seed coat and not in the embryo, resulting in pre‐ and post‐emergence death of seedlings and latent infection. Therefore, quantification of the level of infection by S. tanaceti within seed using a qPCR assay is important for efficient management of the disease. Stagonosporopsis tanaceti completes its life cycle within 12 days after leaf infection through production of pycnidia and can infect every tissue of the pyrethrum plant except the vascular and root tissues. Ray blight epidemics occur in pyrethrum fields through splash dispersal of pycnidiospores between adjacent plants. Besides steam sterilization, thiabendazole/thiram and fludioxonil are effective seed‐treating chemicals in controlling S. tanaceti before planting begins. Ray blight is currently managed in the field through the foliar application of strobilurin fungicides in the first 1–2 years of crop establishment. Later on, difenoconazole and multisite specific fungicides in the next 2–3 years during early spring successfully reduce ray blight infestation. Avoiding development of resistance to fungicides will require more sustainable management of ray blight including the development and deployment of resistant cultivars.

Morphological characterization of Ampelomyces spp., a hyperparasite of Bhendi (Abelmoschus esculentus (L.) Moench) powdery mildew

Ampelomyces is a naturally occurring hyperparasite on powdery mildews. Survey was conducted in major bhendi (Abelmoschus esculentus (L.) Moench) growing districts of Tamil Nadu during June 2014 to assess the incidence of powdery mildew and to collect different isolates of Ampelomyces spp. The results of the survey revealed that the disease incidence ranged from 15.54 to 63.45 %. Ten isolates of Ampelomyces spp. were collect-ed from surveyed areas of powdery mildew. Isolation of Ampelomyces spp. was done from powdery mildew infected bhendi leaf parasitized by Ampelomyces spp. using tissue segment method. All the isolates were identified by their morphological characters. The colour of the colonies in various medium was brownish black to greenish white. Most of the isolates showed radial and fluffy growth pattern with raised growth. The pycnidia of different isolates of Ampelomyces varied in their shape and were mostly ovoid, ellipsoid, cylindrical, pyriform to globose in shape. The size of pycnidia varied from 29.2-72.5×22.4-43.1 ?m. The number of pycnidia was found to be more in isolates viz., TNAU-AQ101 and TNAU-AQ103. Pycnidiospores are hyaline, unicellular and guttulate in shape. The pycnidial production was higher in TNAU-AQ101 and TNAU-AQ103. Application of agrochemicals is one of the oldest and most effective methods to manage powdery mildew disease. However, incessant use of these agrochemicals has many demerits such as development of resistance to pathogens, residual toxicity and environmental pollution. Hence, search for an alternative means for disease management is envisaged. The genus Ampelomyces are the major antagonists as an alternative of Erysiphales fungi being a significant group of phytopathogens.

Production of Pycnidia and Conidia by Guignardia bidwellii, the Causal Agent of Grape Black Rot, as Affected by Temperature and Humidity.

Black rot, caused by the fungus Guignardia bidwellii, is a polycyclic disease affecting grape leaves and berries. In environmentally controlled experiments and in a 3-year field study, the effects of temperature and relative humidity (RH) were assessed on the following growth parameters of G. bidwellii: (i) formation of pycnidia and cirri in grape leaf lesions, (ii) production and germination of conidia, and (iii) length of the period between lesion appearance and pycnidia production. Pycnidia were produced between 5 and 35°C and at 90 to 100% RH but more pycnidia were produced between 20 and 30°C. No pycnidia were produced at RH < 90%. The first pycnidia were produced in approximately 2 days after lesion appearance at ≥20°C and in 8 days at 5°C; pycnidia continued to be produced on the same lesion for 5 to 16 days after lesion appearance, depending on the temperature. Models were developed to describe the effect of temperature and RH on pycnidia production, accounting for 95 and 97% of variability, respectively. Cirri were extruded only between 15 and 35°C and mainly at 100% RH. Field experiments confirmed that pycnidia are produced for several days on a leaf lesion and that the length of the period between lesion appearance and pycnidia production depends on temperature. Overall, the findings showed that production of conidia requires high humidity; under field conditions, some hours at high humidity, which usually occur at nighttime, rather than constant high humidity may be sufficient.

Dissecting the role of histidine kinase and HOG1 mitogen-activated protein kinase signalling in stress tolerance and pathogenicity of Parastagonospora nodorum on wheat.

The HOG1 mitogen-activated protein kinase (MAPK) pathway is activated through two-component histidine kinase (HK) signalling. This pathway was first characterized in the budding yeast Saccharomyces cerevisiae as a regulator of osmotolerance. The fungus Parastagonospora nodorum is the causal agent of septoria nodorum blotch of wheat. This pathogen uses host-specific effectors in tandem with general pathogenicity mechanisms to carry out its infection process. Genes showing strong sequence homology to S. cerevisiae HOG1 signalling pathway genes have been identified in the genome of P. nodorum. In this study, we examined the role of the pathway in the virulence of P. nodorum on wheat by disrupting putative pathway component genes: HOG1 (SNOG_13296) MAPK and NIK1 (SNOG_11631) hybrid HK. Mutants deleted in NIK1 and HOG1 were insensitive to dicarboximide and phenylpyrrole fungicides, but not a fungicide that targets ergosterol biosynthesis. Furthermore, both Δnik1 and Δhog1 mutants showed increased sensitivity to hyperosmotic stress. However, HOG1, but not NIK1, is required for tolerance to elevated temperatures. HOG1 deletion conferred increased tolerance to 6-methoxy-2-benzoxazolinone, a cereal phytoalexin. This suggests that the HOG1 signalling pathway is not exclusively associated with NIK1. Both Δnik1 and Δhog1 mutants retained the ability to infect and cause necrotic lesions on wheat. However, we observed that the Δhog1 mutation resulted in reduced production of pycnidia, asexual fruiting bodies that facilitate spore dispersal during late infection. Our study demonstrated the overlapping and distinct roles of a HOG1 MAPK and two-component HK signalling in P. nodorum growth and pathogenicity.

Sporulation rate in culture and mycoparasitic activity, but not mycohost specificity, are the key factors for selecting Ampelomyces strains for biocontrol of grapevine powdery mildew (Erysiphe necator)

To develop a new biofungicide product against grapevine powdery mildew, caused by Erysiphe necator, cultural characteristics and mycoparasitic activities of pre-selected strains of Ampelomyces spp. were compared in laboratory tests to the commercial strain AQ10. Then, a 2-year experiment was performed in five vineyards with a selected strain, RS1-a, and the AQ10 strain. This consisted of autumn sprays in vineyards as the goal was to reduce the number of chasmothecia of E. necator, and, thus, the amount of overwintering inocula, instead of targeting the conidial stage of the pathogen during spring and summer. This is a yet little explored strategy to manage E. necator in vineyards. Laboratory tests compared the growth and sporulation of colonies of a total of 33 strains in culture; among these, eight strains with superior characteristics were compared to the commercial product AQ10 Biofungicide® in terms of their intra-hyphal spread, pycnidial production, and reduction of both asexual and sexual reproduction in E. necator colonies. Mycoparasitic activities of the eight strains isolated from six different powdery mildew species, including E. necator, did not depend on their mycohost species of origin. Strain RS1-a, isolated from rose powdery mildew, showed, together with three strains from E. necator, the highest rate of parasitism of E. necator chasmothecia. In field experiments, each strain, AQ10 and RS1-a, applied twice in autumn, significantly delayed and reduced early-season development of grapevine powdery mildew in the next year. Therefore, instead of mycohost specificity of Ampelomyces presumed in some works, but not confirmed by this study, the high sporulation rate in culture and the mycoparasitic patterns became the key factors for proposing strain RS1-a for further development as a biocontrol agent of E. necator.

Use of systems analysis to develop plant disease models based on literature data: grape black-rot as a case-study

The available knowledge on black-rot of grape was retrieved from literature, analyzed, and synthesized to develop a mechanistic model of the life cycle of the pathogen (Guignardia bidwelii) based on the systems analysis. Three life-cycle compartments were defined: (i) production and maturation of inoculum in overwintered sources (i.e., ascospores from pseudothecia and conidia from pycnidia in berry mummies and cane lesions); (ii) infection caused by ascospores and conidia; and (iii) disease onset and production of secondary inoculum. An analysis of published, quantitative information was conducted to develop a mechanistic model driven by weather and vine phenology; equations were developed for ascospore and conidial maturation in overwintered fruiting bodies, spore release and survival, infection occurrence and severity, incubation and latency periods, onset of lesions, production of pycnidia, and infectious periods. The model was then evaluated for its ability to represent the real system and its usefulness for understanding black-rot epidemics by using three typical epidemics. Finally, weaknesses in our knowledge are discussed. Additional research is needed concerning the influence of wetness duration and temperature on infection by ascospores, production dynamics of pycnidia and conidia in black-rot lesions, and the dynamics of conidia exudation from pycnidia.

Sources and Availability of Inoculum and Seasonal Survival of Sphaeropsis pyriputrescens in Apple Orchards.

Sphaeropsis pyriputrescens is the cause of Sphaeropsis rot, a recently reported postharvest fruit rot disease of apple. Infection of apple fruit by the fungus is believed to occur in the orchard, and symptoms develop during storage or in the market. S. pyriputrescens also is the cause of a twig dieback and canker disease of apple and crabapple trees. To determine sources of pathogen inoculum in the orchard, twigs with dieback and canker symptoms, dead fruit spurs, dead bark, and fruit mummies on the trees were collected and examined for the presence of pycnidia of S. pyriputrescens. To monitor inoculum availability during the growing season from early May to early November, dead fruit spurs or twigs from Fuji trees, and twigs with dieback from crabapple trees (as a source of pollen for apple production) in a Fuji orchard as well as dead fruit spurs and dead bark from Red Delicious trees in a Red Delicious orchard were sampled periodically and examined for the presence and viability of pycnidia of S. pyriputrescens. To determine seasonal survival and production of pycnidia of the fungus on twigs, apple twigs were inoculated in early December, sampled periodically for up to 12 months after inoculation, examined for the presence of pycnidia, and subjected to isolation of the fungus from diseased tissues to determine its survival. Pycnidia of S. pyriputrescens were observed on diseased twigs, dead fruit spurs and bark, and mummified fruit on both apple and crabapple trees, suggesting that these tissues were the sources of inoculum for fruit infection in the orchard. With the combined observations from two orchards during three growing seasons, viable pycnidia of the fungus were present throughout the year and observed in 50 to 100% of the Fuji trees, >90% of crabapple trees, and 0 to 50% of the Red Delicious trees. S. pyriputrescens was recovered from diseased tissues of inoculated twigs at all sampling times up to 12 months after inoculation. The results suggest that S. pyriputrescens can survive as mycelium in diseased twigs in north-central Washington State and that availability of viable S. pyriputrescens pycnidia is unlikely a limiting factor for infection of apple fruit in the orchard leading to Sphaeropsis rot during storage.

In vitro growth of some species of Ascochyta Lib.

Abstract Fungi from the genus Ascochyta are generally facultative saprotrophs, which cause diseases in both monocots and dicots. Over 1 000 species belonging to this genus have been identified, 18 of which infect monocot plants from the family Poaceae. This study analyses the effects of temperature and light on the growth of selected fungi which infect monocots (A. agrostidis, A. avenae, A. brachypodii, A. desmazieri, A. digraphidis, A. ducis-aprutii, A. festucae, A. graminea, A. hordei, A. hordei var. americana, A. hordei var. europea, A. hordei var. hordei, A. melicae, A. phleina, A. skagwayensis, A. sorghi, A. stipae, A. zeicola), grown on three types of media; Potato Dextrose Agar (PDA), Coon’s agar (CN) and oatmeal agar (OMA). The fastest growth among the analyzed fungi at low temperatures was found in Ascochyta melicae, while at high temperatures it was A. zeicola. The fastest in vitro growth (average of all fungi) was observed on CN medium at 20°C (3.4 mm/day), while the lowest on OM medium at 5°C (1.0 mm/day). Radial mycelial growth in dark and the light conditions varied. On average, all isolates grew faster in the dark (3.1 mm/day) than in the light (1.9 mm/day). The greatest effect on the production of pycnidia was found for the isolates. Variation in growth and production of pycnidia depended on temperature, medium and lighting for fungi from the genus Ascochyta infecting monocots. Such variation indicates a potential occurrence of these fungi in different environments.

Conidial morphology changes in four Phyllosticta species

Production of pycnidia and conidia by Phyllosticta theacearum, Phyllosticta sp. (ex Euonymus) and Phyllosticta sp. (ex Portulaca) (grown on PDA), and P. cruenta (from specimens) was studied. On PDA, conidial appendages of Phyllosticta sp. (ex Portulaca) disappeared completely after 16 days, of P. theacearum after 29 days, and of Phyllosticta sp. (ex Euonymus) after 14 days. In slide preparations mounted in water, appendages of all four species persisted; the appendages of P. cruenta and Phyllosticta sp. (ex Euonymus) became 2–4 times longer within 30 minutes, while those of Phyllosticta sp. (ex Portulaca) and P. theacearum remained unchanged.

First Report of Dieback on Hybrid Rhododendrons Caused by Neofusicoccum luteum and N. parvum in Spain.

During the conducting of Phytophthora ramorum surveys at Galician public parks (northwestern Spain) in 2010, established Rhododendron spp. plants were observed to be exhibiting leaf spots and necrosis, shoot blight, and cankers and dieback of shoots and branches. Branches and leaves of affected rhododendrons contained pseudothecia with bitunicate asci and hyaline pseudoparaphyses, and pycnidia were observed within the same stromatic masses. Symptomatic samples were disinfested in 0.5% sodium hypochlorite for 3 min. Tissues were cut from the margin of lesions, placed onto malt extract agar amended with streptomycin (25 μg ml-1), and incubated at 25°C in the dark. Cultures displaying morphological characteristics associated with Botryosphaeriaceae species were subcultured on 2% water agar with sterilized Pinus pinaster needles as a substrate and incubated at 25°C under near-UV light to encourage pycnidial production (1). Single conidial cultures gave rise to two distinct colonies on potato dextrose agar (PDA) at 25°C. In type 1, isolates produced a sparse, aerial mycelium and a characteristic yellow pigment that was more intense after 3 days, thereafter becoming violaceous and gradually turning dark gray. Growth occurred in the range of 4 to 38°C with an optimum at 29°C. Conidia were hyaline, fusiform, aseptate, thin walled, and averaged 21.1 (14.3 to 25.0) × 5.7 (4.3 to 6.8) μm with a length/width (L/W) ratio of 3.7 ± 0.4 (n = 100). On the basis of these characteristics, isolates were identified as Neofusicoccum luteum (1,3). Colonies of type 2 produced a dense, white-to-yellowish mycelium that rapidly became gray followed by marked diurnal zonation. Mycelial growth occurred in the range of 6 to 38°C with an optimum at 29 to 30°C. Conidia were hyaline, elliptical or fusiform, aseptate, thin walled, and averaging 18.3 (14.1 to 20.7) × 5.8 (4.6 to 7.0) μm with a L/W ratio of 3.2 ± 0.4 (n = 100). These isolates were identified as N. parvum (1,2). Identity was confirmed by DNA sequences analysis of internal transcribed spacer (ITS) regions. Comparison of the sequences of type 1 and 2 showed 100% homology with N. luteum and N. parvum (GenBank Accession Nos. EU673311 and GU251146, respectively). Representative sequences were deposited at GenBank (Accession Nos. HQ197352 and HQ197351). Pathogenicity of each isolate of N. luteum and N. parvum was confirmed by inoculating four 3-year-old Rhododendron spp. seedlings grown in pots. Shallow cuts were made in three branches of each plant. A colonized 6-mm agar plug, removed from the margin of an actively growing colony, was inserted beneath the flap and sealed with Parafilm. Four control seedlings received only sterile PDA agar plugs. Plants were maintained at 26°C and 70% humidity for 21 days. Inoculated plants began showing symptoms after 3 days. Necrosis progressed quickly and bidirectionally from the wound, resulting in death of leaves and wilting of shoots. N. luteum and N. parvum were reisolated from all inoculated plants but not from the controls. To our knowledge, this is the first report of N. luteum and N. parvum on Rhododendron spp. in Spain.