Formation Of Pycnidia Research Articles

Biocontrol of citrus melanose Diaporthe citri by Bacillus subtilis M23

Citrus melanose, caused by Diaporthe citri, is one of the most prevalent and important fungal diseases of citrus crops globally. However, the overuse of chemical fungicides for disease control has an adverse impact on citrus production. In contrast, biological control agents (BCAs) are environmentally friendly and have become essential tools for plant disease control. In the present study, Bacillus subtilis M23, a strain isolated from healthy citrus leaves, significantly suppressed the mycelial growth of D. citri, the causal agent of citrus melanose, in vitro. The fermentation broth of B. subtilis M23 also exhibited good antifungal activity against D. citri on citrus plants in greenhouse and field experiments. Lipopeptides (LPs) produced by M23 exhibited excellent antifungal activity against D. citri, with a 50 % effective concentration (EC50) of approximately 1.00 and 0.28 μg/mL for inhibition of mycelial growth and conidial germination, respectively. LP biosynthesis genes were identified in the M23 genome using a PCR assay. Notably, LP extracts significantly reduced the formation of pycnidia and ATP biosynthesis in D. citri. Comparative transcriptome analysis revealed significant differences in the expression of genes associated with fungal vacuole, oxidoreductase activity and endopeptidase activity in D. citri treated with B. subtilis M23. Both RNA-seq and RT-qPCR analysis provided similar results on the expression of selected genes. Collectively, our data provided convincing data supporting the biocontrol potential of B. subtilis M23 for the management of citrus melanose.

Diaporthe ueckeri causing cassava root rot in Pernambuco, Brazil

Cassava root rot is a highly destructive disease that causes great damage in Brazil. Identification of the causal agents is essential for the management of this disease. Roots of cassava plants (Manihot esculenta Crantz) with symptoms of root rot were collected from a cassava crop in the municipality of Garanhuns, Pernambuco State, Brazil. This study aimed to identify a Diaporthe species isolated from cassava root rot in Brazil and evaluate pathogenicity in cassava plants and roots. At 7 days after inoculation (DAI), tuberous roots showed necrotic lesions, a soft appearance, and pink color in the center and brown to greenish at the edges, while seedlings showed root rot, wilting, yellowing, drying out, and leaf fall at 66 DAI in the soil. All plants in the control group were asymptomatic. After reisolations and confirmation of Koch's postulates, the causal agent was conclusively identified as Diaporthe ueckeri through the multilocus phylogenetic analyses involving the ITS, TEF1-α, TUB2, CAL, and HIS genes. The results showed that the morphological characteristics, such as colonies with abundant aerial mycelium, formation of pycnidia and alpha conidia, were consistent with those described for species of the genus Diaporthe. This is the first report of D. ueckeri causing cassava root rot in Brazil and worldwide.

First report of Diplodia bulgarica causing black canker on apple in California.

California is the sixth largest apple-producing state in the United States with a production that reached 4,654 ha in 2021. During the late winter of 2023, black canker symptoms were observed on branches of 'Gravenstein' apple (Malus domestica) in two commercial orchards in Sonoma County, California. The prevalence of symptomatic trees ranged from 10 to 30%. External symptoms included charcoal looking-cankers with the bark peeling off from the primary and secondary branches. Internally, cankers were dark brown in color with a hard consistency. Pycnidia were observed on the surface of older cankers. Fungal isolations were performed from disinfected (70% ethanol, 30 s) symptomatic branch samples (n = 15). Small wood pieces (5 mm length) were taken from the margin of diseased and healthy tissues, and placed on potato dextrose agar acidified with 92% lactic acid at 0.5 mL per liter (APDA). Plates were incubated at room temperature (20-22 °C) for 7 days. Colonies of Botryosphaeriaceae species (Phillips et al. 2013) (n = 12) were consistently recovered and pure cultures were obtained by transferring a single hyphal tip onto fresh APDA. Colonies were light gray with irregular margins. To induce pycnidia formation, two isolates (UCD11350 and UCD11351) were grown on pistachio leaf agar for 21 days. Conidia (n = 50) were thick-walled and ovoid in shape, initially hyaline, then turned pale brown and dark brown at maturity, and some of them became 1-septate, ranging from 18.9 to 24.0 (21.9) × 11.5 to 14.7 (13.4) µm. Isolates were identified by sequencing a partial region of the beta-tubulin (tub2) gene using the primers Bt2a/Bt2b (Glass and Donaldson 1995). BLAST searches on NCBI GenBank revealed 99.5 % identity with the Diplodia bulgarica ex-type (CBS 1245254). To confirm the identity, the rRNA internal transcribed spacer (ITS) and the translation elongation factor 1-alpha (tef1) were also sequenced using ITS5/ITS4 (White et al. 1990), and EF1-688F/EF1-1251R (Alves et al. 2008), respectively. A maximum parsimony multi-locus phylogenetic analysis clustered Californian isolates with reference strains of D. bulgarica. Sequences were deposited in GenBank (nos. OR631209 to OR631210, OR637361 to OR637362, OR637363 to OR637364 for ITS, tub2, and tef1, respectively). Pathogenicity tests were conducted on 2 to 3-year-old branches (n = 5) of over 20-year-old trees by inserting a 5-mm segment of a toothpick, completely colonized with each of the two isolates mentioned above, into a 1-mm-diameter hole made with a sterile drill bit. The same number of branches where mock inoculated with a non-colonized toothpick as negative control. The experiment was performed twice. After ten weeks, inoculations resulted in dark brown necrotic lesions that ranged from 54.0 to 59.8 mm in length. Negative controls remained asymptomatic. Koch's postulates were fulfilled by successfully recovering the isolates from the lesion margins, which were confirmed by morphology. Diplodia bulgarica was first described affecting M. sylvestris in Bulgaria (Phillips et al. 2012), and then detected on M. domestica causing cankers in Iran (Abdollahzadeh 2015), India (Nabi et al. 2020), Germany (Hinrichs-Berger al. 2021) and Türkiye (Eken 2021). The pathogen was also identified causing postharvest fruit rot (Eken 2022). To our knowledge, this is the first report of D. bulgarica causing branch canker on apple in California, which provides important information for developing detection and control strategies.

Characterization of Durum Wheat Resistance against Septoria Tritici Blotch under Climate Change Conditions of Increasing Temperature and CO2 Concentration

Wheat interactions against fungal pathogens, such as Zymoseptoria tritici, are affected by changes in abiotic factors resulting from global climate change. This situation demands in-depth knowledge of how predicted increases in temperature and CO2 concentration ([CO2]) will affect wheat—Z. tritici interactions, especially in durum wheat, which is mainly grown in areas considered to be hotspots of climate change. Therefore, we characterized the response of one susceptible and two resistant durum wheat accessions against Z. tritici under different environments in greenhouse assays, simulating the predicted conditions of elevated temperature and [CO2] in the far future period of 2070–2099 for the wheat-growing region of Córdoba, Spain. The exposure of the wheat—Z. tritici pathosystem to elevated temperature reduced disease incidence compared with the baseline weather conditions, mainly affecting pathogen virulence, especially at the stages of host penetration and pycnidia formation and maturation. Interestingly, simultaneous exposure to elevated temperature and [CO2] slightly increased Z. tritici leaf tissue colonization compared with elevated temperature weather conditions, although this fungal growth did not occur in comparison with baseline conditions, suggesting that temperature was the main abiotic factor modulating the response of this pathosystem, in which elevated [CO2] slightly favored fungal development.

Understanding Environmental and Physiological Factors Affecting the Biology of Diaporthe ilicicola, the Fungus Causing Latent Fruit Rot in Winterberry.

Fruit rot in winterberry is associated with a complex of fungal pathogens. Among them, Diaporthe ilicicola plays a unique role by infecting flowers at bloom, resulting in symptom development in mature fruit. This research aimed to identify at what stage of maturation Ilex fruit can develop disease symptoms and correlate changes in fruit physiology (sugar and phenolic content) and environment (temperature and light intensity) with disease incidence. Correlation data informed in vitro studies testing the ability of putative factors to alter growth of D. ilicicola and select opportunistic fungi within the fruit rot complex: Alternaria alternata, Colletotrichum fioriniae, and Epicoccum nigrum. Results indicated that Ilex fruit do not develop symptoms until 81 to 108 days after inoculation. Temperature and fruit phenolic content were negatively correlated with disease incidence, while fruit sugar concentration and light intensity were positively correlated. In vitro assays revealed that sugar concentration had no effect on the growth of D. ilicicola, but increased light intensity increased hyphal growth and pycnidium formation. Additionally, phenolics extracted from fruit inhibited spore germination in A. alternata, induced secondary conidiation in C. fioriniae, and late season phenolic extracts increased hyphal melanization and pycnidial formation in D. ilicicola. Finally, drops in field temperatures, when replicated in vitro, resulted in a decrease in hyphal growth and spore germination for all fungi. These results suggest that changes in Ilex fruit phenolics during maturation and the increased exposure to light following defoliation may play a role in symptom development by altering D. ilicicola growth within the fruit.

Sas3-mediated histone acetylation regulates effector gene activation in a fungal plant pathogen.

Pathogen infections require the production of effectors that enable host colonization. Effectors have diverse functions and are only expressed at certain stages of the infection cycle. Thus, effector genes are tightly regulated by several mechanisms, including chromatin remodeling. Here, we investigate the role of histone acetylation in effector gene activation in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that lysine acetyltransferases (KATs) are essential for the spatiotemporal regulation of effector genes. We show that the KAT Sas3 is involved in leaf symptom development and pycnidia formation. Importantly, our results indicate that Sas3 controls histone acetylation of effector loci and is a regulator of effector gene activation during stomatal penetration. Overall, our work demonstrates the key role of histone acetylation in regulating gene expression associated with plant infection.

First Report of Neofusicoccum mediterraneum and Neofusicoccum parvum Causing Pine Ghost Canker on Pinus spp. in Southern California.

Pinus eldarica, P. halepensis and P. radiata are important conifer species native to Mediterranean regions that are cultivated in the southwestern United States for landscaping (Phillips and Gladfelter, 1991; Chambel et al., 2013). Among them, Monterey pine (P. radiata) is native to restricted areas of California and Mexico, but it is extensively grown for timber production in other countries, especially in the Southern Hemisphere (Rogers, 2004). From 2018 to 2022, severe dieback and cankers have been detected on more than 30 mature pines of the three species within a 40-ha urban forest in Orange County, Southern California. Symptoms initiate on the lower portion of the canopy and advance into the crown, leading to quick dieback and, in some cases, to tree death. Cross sections of affected branches revealed wedged cankers with irregular, indistinct margins, and cryptic discoloration (i.e., "ghost cankers"). Pycnidia were observed on the surface of each bark scale of branches with advanced infections. Two morphotypes of Botryosphaeriaceae colonies (n = 34 isolates) were recovered consistently from more than 90% of the symptomatic pines. Two isolates per morphotype were grown on pistachio leaf agar (Chen et al., 2014) for 14 days to induce pycnidia formation. Conidia (n = 50) were hyaline, thin-walled and fusoid to ellipsoidal in shape, ranging from 16.1 to 27.9 (22.6) × 5.4 to 8.2 (6.8) µm for the first morphotype and 11.5 to 20.4 (16.3) × 4.8 to 8.6 (6.3) µm for the second morphotype. The rDNA internal transcribed spacer (ITS), beta-tubulin (tub2), and translation elongation factor 1-alpha (tef1-α) partial gene regions were amplified and sequenced using the primers ITS5/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995), and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. A multi-locus phylogenetic analysis revealed that isolates UCD9433 and UCD10439 clustered with the ex-type strain of Neofusicoccum mediterraneum (CBS:113083), and isolates UCD9161 and UCD9434 grouped with N. parvum (CMW:9081). Sequences were submitted to GenBank (nos. OP535391 to OP535394 for ITS, OP561946 to OP561949 for tef1-α, and OP561950 to OP561953 for tub2). Pathogenicity tests were performed with above-mentioned isolates on 20-mm-diameter healthy branches of mature Monterey pines (n = 10, 14 years old) located in a research field at UC Davis. Isolates were grown for 7 days on potato dextrose agar and inoculated in the internode area by removing a 5-mm-diameter disk of the bark with a sterile cork borer and placing a 5-mm-diameter mycelial plug. Controls were mock-inoculated with sterile agar plugs, and the experiment was performed twice. After three months, inoculations resulted in vascular lesions that ranged from 20.6 to 49.7 (32.7) mm with N. mediterraneum and from 13.5 to 71.0 (33.6) mm with N. parvum, and the same pathogens were reisolated (70 to 100% recovery). Controls remained symptomless and no botryosphaeriaceous colonies were recovered. Both N. mediterraneum and N. parvum are polyphagous pathogens associated with multiple woody plant hosts (Phillips et al., 2013). Previously, only N. parvum has been associated with pine cankers in Iran, however, the pine species was not indicated (Abdollahzadeh et al., 2013). The detection of these pathogens in urban forests raises concerns of potential spillover events to other forest and agricultural hosts in Southern California. To our knowledge, this is the first report of N. mediterraneum and N. parvum causing Pine Ghost Canker on P. eldarica, P. halepensis and P. radiata.

First Report of Diaporthe eres Causing Leaf Spot on Pseudocydonia sinensis in China.

Pseudocydonia sinensis is a Chinese ornamental plant with great landscaping value. Its fruit is additionally used for medicinal purposes (Lim 2012). In June 2020, a leaf spot disease was observed in the campus of Nanjing Forestry University (32°04'34.53″N 118°48'42.06″E). The symptoms began with irregular red-brown spots, which gradually enlarged, extended and overlapped, with an incidence of 85% (29/34 trees). Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of tissues. Pure cultures were obtained by monosporic isolation.The representative isolate NJMG 5-7 was used for morphological and molecular characterization. The growing fungal colony on PDA was initially white, but gradually turned grey. Pycnidia formation was observed on PDA supplemented with alfalfa stems. The pycnidia produced two different types of conidia, α and β, which ooze out in yellow creamy mucilaginous masses. Conidiophores were hyaline, cylindrical and smooth, 16.8 to 33.1 × 1.5 to 2.6 μm (n=30). Conidiogenous cells were 13.6 to 29.3 × 1.5 to 2.7 μm (n=30). The α-conidia were, unicellular, hyaline elliptical or fusiform, bi-guttulate, 6.5 to 9.2 × 1.8 to 3.3 µm (n = 50). The β-conidia were hyaline, aseptate, without guttules, filiform, curved, with obtuse ends, 12.5 to 25 × 1.0 to 1.8 µm (n = 50). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, and calmodulin (CAL), translation elongation factor 1 alpha (EF1-a), and beta-tubulin genes (TUB) were amplified from isolate NJMG 5-7 with primers ITS1/ITS4 (White et al. 1990), CAL-228F/CAL-737R (Carbone & Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (OP223050 for ITS, OP252809 for CAL, OP252807 for EF1-a, and OP252808 for TUB). A BLAST search of GenBank showed that ITS, CAL, EF1-a and TUB sequences of NJMG 5-7 were similar to those of D. eres CBS 138594 (99% identity), AR5193 (99%), AR5193 (99%) and MG281193 (100%), respectively. The morphological and molecular results identified the isolate as D. eres (Feng et al. 2015). To fulfill Koch's postulates, a pathogenicity test was conducted using three P. sinensis plants. Six leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of D. eres from a 3-day-old culture grown on PDA. Inoculations with sterile PDA plugs on different leaves of the same tree were used as controls. All inoculated leaves were enclosed in plastic bags together with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags to keep moisture conditions. Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and identified from its morphology, thus fulfilling Koch's postulates. This fungus can cause a variety of diseases. To our knowledge, this is the first report of D. eres causing leaf spots on P. sinensis in the world. These findings provide a foundation for future studies on the epidemiology and control of this newly emerging disease.

In vitro and in vivo activity of chemical fungicides and a biofungicide for the control of wood diseases caused by botryosphaeriales fungi in apple and pear

Dieback and canker disease caused by species of Botryosphaeriales affect the productivity of pome fruit orchards. In this study, we evaluated the efficacy of fungicides and biofungicide for controlling canker and dieback diseases in apple and pear using in vitro and field trials. Isolates of Diplodia seriata, D. mutila, and Dothiorella omnivora from apple, and of D. seriata and Aplosporella aquifolii from pear were used. Cercobin® (thiophanate-methyl), followed by Bellis® (pyraclostrobin + boscalid), and Nativo® (tebuconazole + trifloxystrobin) inhibited mycelial growth, conidial germination and pycnidia formation in vitro in all fungal isolates. T. harzianum produced mycelial growth inhibition and mycoparasitism in dual culture assays. Thiophanate-methyl and T. harzianum fully inhibited pycnidia production, while pyraclostrobin + boscalid and tebuconazole + trifloxystrobin were most effective against conidia germination. In the orchard assays, protective sprays of the fungicides on pruning wounds reduced incidence by up to 20%, while T. harzianum and tebuconazole + trifloxystrobin reduced the lesion severity up 37 and 41%, respectively. This study represents the first evaluation of fungicides and biofungicide effectiveness for the control of canker and dieback diseases of apples and pears in Argentina.

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici.

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes (Stb), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici. Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q-mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q-mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb-mediated resistances.

Canker and leaf scorch on olive (Olea europaea L.) caused by Neoscytalidium dimidiatum in Turkey

In a recent survey of olive groves in the Southeast Anatolia Region of Turkey, a new and serious decline of olive trees, beginning with foliar scorching and then dieback of twigs, branches, and even whole trees, was observed for the first time. In more advanced stages of the disease, necrosis and cankers were observed on the bark of the trunk, branches, and twigs. Isolations from symptomatic tissues from multiple cultivars in diverse locations yielded Neoscytalidium dimidiatum, which were identified using ITS, tef1, and tub2 loci of genomic DNA, in combination with morphological data. In vitro studies showed that conidial germination, hyphal growth, and pycnidia formation of the pathogen were positively correlated with elevated temperatures. Wild type olive “Delice”, “Edincik Su”, and “Memecik” seemed like the most resistant cultivars on which disease severity values were the lowest among 14 screened olive cultivars in two experimental orchards under natural conditions. Pathogenicity tests showed that “Gemlik” was the most susceptible cultivar showing the largest cankers and extensive scorch lesions. Isolates caused canker but not leaf scorch on O. europaea cultivars “Arbequina” “Halhalı”, “Manzanilla”, “Nizip Yağlık”, and “Saurani”. Neoscytalidium isolates are likely to have a negative impact on the health of diverse olive groves, which are primarily confined to Mediterranean-type climatic regions. These findings suggest an increased risk of infection in environments with increasing temperatures, as is common in the Southeast Anatolia Region of Turkey. The information gathered in this study will be used to examine the disease's epidemiology and establish disease control initiatives. This is also the first report of N. dimidiatum infecting O. europaea in the world.

A Laminarin-Based Formulation Protects Wheat Against Zymoseptoria tritici via Direct Antifungal Activity and Elicitation of Host Defense-Related Genes.

The present study aimed to evaluate the potential of the laminarin-based formulation Vacciplant to protect and induce resistance in wheat against Zymoseptoria tritici, a major pathogen on this crop. Under greenhouse conditions, a single foliar spraying of the product 2 days before inoculation with Z. tritici reduced disease severity and pycnidium density by 42 and 45%, respectively. Vacciplant exhibited a direct antifungal activity on Z. tritici conidial germination both invitro and in planta. Moreover, it reduced in planta substomatal colonization as well as pycnidium formation on treated leaves. Molecular investigations revealed that Vacciplant elicits but did not prime the expression of several wheat genes related to defense pathways, including phenylpropanoids (phenylalanine ammonia-lyase and chalcone synthase), octadecanoids (lipoxygenase and allene oxide synthase), and pathogenesis-related proteins (β-1,3-endoglucanase and chitinase). By contrast, it did not modulate the expression of oxalate oxidase gene involved in the reactive oxygen species metabolism. Ultrahigh-performance liquid chromatography-mass spectrometry analysis indicated limited changes in leaf metabolome after product application in both noninoculated and inoculated conditions, suggesting a low metabolic cost associated with induction of plant resistance. This study provides evidence that the laminarin-based formulation confers protection to wheat against Z. tritici through direct antifungal activity and elicitation of plant defense-associated genes.

Identification of Colletotrichum liriopes as the Causative Agent of Anthracnose in Buckwheat (Fagopyrum esculentum) in China.

Buckwheat (Fagopyrum esculentum), belonging to the Polygonaceae family, is one of the most important "functional food" crops in China. In fall 2020, buckwheat plants grown in field exhibiting stem canker symptoms were found in Tongxin county, Ningxia province, China. Symptoms included stem canker, dieback and extensive vascular discoloration. Cankers were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips, pycnidia formation was also observed within the cankers. Disease incidence was approximately 30% and moderate to high severity across the field. Symptomatic tissues were cut into 1-2 cm pieces, surface sterilized (75% ethanol for 30 s and 0.1% NaClO for 2 min) and washed four times with sterile distilled water, dried in sterile filter paper for 3 times, and placed on potato dextrose agar (PDA) at 25 ℃. Fluffy mycelium was visible for all isolates after 48 h of incubation. Twenty-five single isolates were hyphal-tip purified on PDA. Six representative isolates were used for further study. The fungal colonies on PDA were flat with an entire margin, gray aerial hyphae, light brown pigmentation, appressed slimy mycelium within which numerous brown-black perithecia formed. Colonies on oatmeal agar (OA) were flat, with flocculent mycelium, conidiomata and conidia and the reverse side was black to smoke-grey. Sparse brown-black perithecia were observed within the mycelium. Conidia were hyaline, one-celled, smooth-walled, rarely finely verruculose, aseptate, slightly curved, both sides gradually tapering towards the round to slightly acute apex and truncate base, measured (15.7-23.7) µm (length) × (2.8-5.7) µm (width), (avg. 20.2 µm×4.2 µm, n=100). Genomic DNA was extracted from the same six isolates, the internal transcribed spacer (ITS) region and the genes encoding beta-tubulin (TUB), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 (HIS3) were independently amplified with primers V9G/ITS4, T1/Bt-2b, CHS-354R/CHS-79F, GDF1/GDR1 and CYLH3F/CYLH3R, respectively (Damm et al., 2019). Sequences for all six isolates were identical. The sequences of the representative isolate 9J1 were deposited in GenBank (accession nos. MW819604, MW836580, MW836577, MW836578 and MW836579). The results of BLASTn showed that the ITS, TUB, CHS, GAPDH and HIS3 sequences of 9J1 were greater than 98% (555/557bp, 477/486bp, 258/259bp, 242/248bp and 339/345bp, respectively) identical to those of Colletotrichum liriopes (GenBank: MT645674 (ITS), GU228098 (TUB), MT663546 (CHS), MH291255 (GAPDH) and MH292811 (HIS3)). MrBayes phylogenetic analysis using concatenated sequences of ITS, TUB, CHS, GAPDH and HIS3 showed that the isolate clustered monophyletically with strains of C. liriopes. Based on morphological and molecular characteristics, the isolate was identified as C. liriopes. To fulfill Koch's postulates, spores of the isolate 9J1 grown on OA for 14 days were harvested in 0.01% Tween 20, and the suspension were adjusted to 104 spores/ml. Six one-month-old potted plants of buckwheat were inoculated by spraying the spore suspension until run-off. Plants were kept inside a plastic bag for 3 d to maintain high relative humidity and maintained in the greenhouse. Six control plants were sprayed with sterile deionized water and kept under the same conditions. Two weeks after inoculation, all inoculated plants showed stem canker symptoms as described above, whereas control plants remained healthy. The pathogen was successfully reisolated from leaf and stem symptomatic tissue, and identified as C. liriopes based on morphological features and DNA sequence analysis, thereby fulfilling Koch's postulates. C. liriopes has been reported causing anthracnose on Eria coronaria (Yang et al., 2011), Liriope spicata (Chen et al., 2019) in China, Liriope muscari in Mexico (Damm et al., 2009), Rohdea japonica in Korea (Kwon and Kim, 2013) and in the United States (Trigiano et al., 2018). To our knowledge, this is the first report of C. liriopes causing anthracnose on buckwheat worldwide. Occurrence of this disease may represent a significant impact for buckwheat production because this crop is the major agricultural commodity in some parts of China. More studies are needed to understand the epidemiology of this disease and foster disease management programs in China.

The Algal Polysaccharide Ulvan Induces Resistance in Wheat Against Zymoseptoria tritici Without Major Alteration of Leaf Metabolome

This study aimed to examine the ability of ulvan, a water-soluble polysaccharide from the green seaweed Ulva fasciata, to provide protection and induce resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici. Matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis indicated that ulvan is mainly composed of unsaturated monosaccharides (rhamnose, rhamnose-3-sulfate, and xylose) and numerous uronic acid residues. In the greenhouse, foliar application of ulvan at 10 mg.ml–1 2 days before fungal inoculation reduced disease severity and pycnidium density by 45 and 50%, respectively. Ulvan did not exhibit any direct antifungal activity toward Z. tritici, neither in vitro nor in planta. However, ulvan treatment significantly reduced substomatal colonization and pycnidium formation within the mesophyll of treated leaves. Molecular assays revealed that ulvan spraying elicits, but does not prime, the expression of genes involved in several wheat defense pathways, including pathogenesis-related proteins (β-1,3-endoglucanase and chitinase), reactive oxygen species metabolism (oxalate oxidase), and the octadecanoid pathway (lipoxygenase and allene oxide synthase), while no upregulation was recorded for gene markers of the phenylpropanoid pathway (phenylalanine ammonia-lyase and chalcone synthase). Interestingly, the quantification of 83 metabolites from major chemical families using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in both non-infectious and infectious conditions showed no substantial changes in wheat metabolome upon ulvan treatment, suggesting a low metabolic cost associated with ulvan-induced resistance. Our findings provide evidence that ulvan confers protection and triggers defense mechanisms in wheat against Z. tritici without major modification of the plant physiology.

β-Glucosidase VmGlu2 Contributes to the Virulence of Valsa mali in Apple Tree.

The apple tree canker is caused by Valsa mali, which produces major pathogenic factors involving multiple cell wall-degrading enzymes (CWDEs) and toxins. The β-glucosidases are among the main CWDEs, and thus, they play important roles in the virulence of necrotrophic pathogens. However, the specific roles of β-glucosidases in the virulence of V. mlai remain largely unknown. In this study, we identified a β-glucosidase gene, VmGlu2, which was upregulated during the V. mali infection. We found that VmGlu2 protein had high enzyme activity of β-glucosidase using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate, while the VmGlu2 could convert phloridzin to phloretin with the release of glucose. The deletion and overexpression of VmGlu2 showed no effect on vegetative growth, but gene deletion mutants of V. mlai showed significantly reduced pycnidia formation. The gene deletion mutants had lower β-glucosidase activities and toxin levels as compared to the wild-type strain. Therefore, these mutants showed a reduced virulence. Moreover, the overexpression of VmGlu2 did not affect toxin levels, but it significantly enhanced β-glucosidase activities, which resulted in an increased pathogenicity. Thus, we conclude that VmGlu2 is required for the full virulence of V. mali. These results provide valuable evidence to the complex role of CWDEs in the fungal pathogenicity.

Identification and characterization of Coniella javanica causing leaf blight of kenaf (Hibiscus cannabinus) in Malaysia

Kenaf (Hibiscus cannabinus) has been widely grown in Malaysia because of its fiber content and economic value. In October 2019, blight symptoms were observed on kenaf leaves in a plantation in Chuping, Perlis, Malaysia. The symptoms appeared as irregular, yellowish to brownish water-soaked lesions, which expanded and are accompanied by pycnidial formation in concentric rings. At severe stage, the blighted leaves became dried and defoliated. This study aimed to investigate the causal pathogen of the leaf blight of H. cannabinus. Overall, 20 fungal isolates recovered from the blighted leaves of H. cannabinus were identified as Coniella javanica based on their morphological characteristics and DNA sequences of the internal transcribed spacer (ITS) region and translation elongation factor 1-α (TEF1-α) gene. Phylogenetic analysis of combined dataset of the ITS and TEF1-α, congregated the fungal isolates with the reference isolates of C. javanica (CBS 455.68 and GUE-29). The pathogenicity of C. javanica isolates was confirmed through pathogenicity tests. To our knowledge, this is the first report of C. javanica causing the leaf blight of H. cannabinus in Malaysia.

The Effects of Postharvest Treatments and Sunlight Exposure on the Reproductive Capability and Viability of Phyllosticta citricarpa in Citrus Black Spot Fruit Lesions.

Citrus black spot (CBS) is caused by Phyllosticta citricarpa, which is classified as a quarantine organism in certain countries whose concerns are that CBS-infected fruit may be a pathway for introduction of the pathogen. This study evaluated the reproductive capability and viability of P. citricarpa under simulated conditions in which the whole fruit, peel segments, or citrus pulp with CBS lesions were discarded. Naturally infected ‘Midknight’ Valencia orange and ‘Eureka’ lemon fruit, either treated using standard postharvest sanitation, fungicide, and wax coating treatments or untreated, were placed into cold storage for 5 weeks (oranges at 4 °C and lemons at 7 °C). Thereafter, treated and untreated fruit were incubated for a further 2 weeks at conditions conducive for CBS symptom expression and formation of pycnidia. The ability of pycnidia to secrete viable pycnidiospores after whole fruit and peel segments or peel pieces from citrus pulp were exposed to sunlight at warm temperatures (±28 °C) and ±75% relative humidity levels was then investigated. The combination of postharvest treatments and cold storage effectively controlled CBS latent infections (>83.6% control) and pycnidium formation (<1.4% of lesions formed pycnidia), and the wax coating completely inhibited pycnidiospore release in fruit and peel segments. Pycnidiospores were secreted only from lesions on untreated fruit and peel segments and at low levels (4.3–8.6%) from peel pieces from pulped treated fruit. However, spore release rapidly declined when exposed to sunlight conditions (1.4% and 0% after 2 and 3 days, respectively). The generally poor reproductive ability and viability of CBS fruit lesions on harvested fruit, particularly when exposed to sunlight conditions, supports the conclusion that citrus fruit without leaves is not an epidemiologically significant pathway for the entry, establishment, and spread of P. citricarpa.

MADS-Box Transcription Factor ZtRlm1 Is Responsible for Virulence and Development of the Fungal Wheat Pathogen Zymoseptoria tritici

Zymoseptoria tritici is one of the most economically destructive wheat diseases all over the world and is a model fungal plant pathogen within the ascomycetes. In this study, the instrumental role of the ZtRlm1 gene encoding a MADS-box transcription factor (TF) in the infection process of Z. tritici was functionally characterized as these proteins play critical roles in the global gene regulation required for various developmental and physiological processes. Our infection assays showed that ZtRlm1 mutants were attenuated in disease development as a 30 and 90% reduction in chloro-necrotic lesions and pycnidia formation, respectively, were observed in plants inoculated with ZtRlm1 mutant strains demonstrating that ZtRlm1 is a crucial factor playing a significant role in the late stage of infection corresponding with pycnidial formation. Our expression analysis demonstrated that the transcript level of ZtRlm1 is induced at 2 and 20 days post-inoculation, coinciding with pycnidial sporulation. In addition, microscopic analyses showed that branch intensity and biomass production were significantly reduced, indicating that impaired pycnidia formation is a result of impaired differentiation and biomass production in the ZtRlm1 mutants. Furthermore, melanization, a phenomenon required for fruiting body formation, was significantly hampered in ZtRlm1 mutants as they were not melanized under all tested temperature and media conditions. Overall, our data showed that impaired disease development of the ZtRlm1 mutants is mainly due to the significant impact of ZtRlm1 in different cellular processes, including differentiation, branching, fungal biomass production, and melanization, in which identification of downstream genes are of interest to increase our understanding of this pathosystem.

First Report of Leaf Blight on Aucuba japonica Caused by Nothophoma quercina in China

First Report of Leaf Blight on <i>Aucuba japonica</i> Caused by <i>Nothophoma quercina</i> in China

Phoma macrostoma сирек кездесетін онихомикоз қоздырғышының сипаттамасы (син. Didymella macrostoma)

The article presents the results of studying the biological properties of the Phoma macrostomastrain, first described in Kazakhstan as the causative agent of onychomycosis. The authors obtained a pure culture of Phoma macrostoma coelomic, isolated from the affected nail plate of a patient in a narcological dispensary, and studied its biological properties: cultural, morphological, biochemical. Surface cultivation on differential media, microscopy in agar blocks and adhesive tape preparations, molecular genetic identification was carried out. The features of the formation of fungus colony are described, the characteristic morphological structures of the coelomycete, moderate fermentation of carbohydrates and the absence of urease activity are revealed. On the dextrose agar of Sabouraud nutrient medium, by 10-18 days, flat colonies of pale pink colour, with a pinkish-orange tint in the centre, yellowish along the edge, with a raised centre and relief radial stripes, were formed, the reverser was yellow-brown. The appearance of single growth tubes was noted within the first 12 hours, the massive division of single cells and the growth of filamentous filaments - 18 hours after sowing the spores. By the end of the second day, there is a powerful growth and active branching of the mycelium, the appearance of rudiments of pycnidia in the form of seals along the course of mature mycelial filaments, by the end of the third day, the formation of pycnidia and chlamydospores of the brown colour of various shades. Ripe transparent, unicellular, elliptical pycnospores emerge from pycnidia and located in the form of a mucous drop. The strain is characterized by moderate saccharolytic activity in relation to glucose, sucrose, mannose, mannitol, lactose, the almost complete absence of urease activity. Molecular genetic identification by the Sanger method made it possible to identify the Phoma macrostoma strain with 100% certainty and to deposit the nucleotide sequence in GenBank as isolate No. MN701978.1 Didymella macrostoma 104-Kz. Key words: biology of the pathogen, genetic identification, onychomycosis, pycnidia, coelomycetes