Identification, characterization, and sensitivity to phytochemicals of a novel Curvularia species associated with leaf spot disease on Curcuma kwangsiensis.

A leaf spot disease affecting Curcuma kwangsiensis (Zingiberaceae) has been observed in Qinzhou City, Guangxi Province. Infected leaves exhibit yellow-brown spots that progressively expand and eventually lead to leaf death. Curvularia isolates were obtained from the diseased leaves with tissue isolation and single spore purification methods. To accurately identify these isolates, we analyzed their morphological characteristics and phylogenetic relationships using combinations of ITS, GAPDH, and EF-1α gene sequences. Phylogenetic analysis showed that the investigated strains formed a distinct clade separate from other recognized Curvularia species. Furthermore, the strains exhibited differences in conidiophore size and conidia shape/size. Based on phylogenetic studies, morphology, and pathogenicity tests, the pathogen was identified as a new species named Curvularia qinzhouensis. Optimal conditions for mycelial growth were observed at 30 °C and pH 8. The sensitivity of the pathogen to various phytochemicals was also examined. Honokiol, thymol, and citral demonstrated effective antifungal effects, with EC50 values of 6.72 ± 1.75, 25.74 ± 4.30, and 54.24 ± 4.69 µg/ml, respectively. The present investigation provides the first report of leaf spot disease on C. kwangsiensis caused by C. qinzhouensis, and valuable insights for the prevention and control of this disease.

First Report of Leaf Spot Caused by Corynespora cassiicola on Pogostemon cablin in Guangdong, China.

Pogostemon cablin (Blanco) Benth. is a vital source of patchouli oil, utilized in traditional Chinese medicine, Ayurveda, cosmetics, and hygiene products(Swamy et al. 2016). In 2022, a leaf spot disease outbreak on patchouli plants occurred nearly 10 acres in Yunfu, Guangdong (21°2' N; 110°3' E), with an average incidence rate of 50%. Infected leaves initially showed circular spots with tan centers and yellow halos. Within five to ten days, these spots expanded, crossed leaf veins, and became polygonal with rough surfaces. As the disease progressed, the spots merged, darkening the veins and eventually causing leaf loss. Five symptomatic leaves were soaked in 75% ethanol for 10 s, and 2.5% sodium hypochlorite for 30 s, followed by a final rinse in sterile water. These samples were then placed on potato dextrose agar (PDA) plates. After 3-5 days of incubation at 28 °C, the mycelial growth was transferred to fresh PDA plates and purified by isolating the hyphal tip three times. Four of the five isolates had similar morphology and caused leaf spot symptoms upon inoculation. During culture on PDA, these isolates formed colonies with downy, gray-brown mycelium and darker centers. Microscopic examination revealed branched, septate mycelium; solitary, erect, unbranched grayish-brown conidiophores; and club-shaped conidia. Conidia were faint brown, segmented, contained 2 to 15 septa(predominant number being 3 to 4), ranged from 32 to 220 µm in length and 8.4 to 22.4 µm in width (n = 30). The morphology of these isolates was identical to that of Corynespora cassiicola (Ellis 1971). Pathogenicity tests were conducted on 3-month-old seedlings of P. cablin using the conidium infection method. A suspension of conidia (1 × 106 conidia/ml) was prepared from cultures induced to sporulate by 90-minute near-UV exposure followed by 2-day dark incubation at 28 °C, and 30 ml of this suspension was sprayed onto leaves of each seedling. Inoculated plants were incubated at 28 °C in an incubator. Potted plants treated only with sterile water were used as controls. Each treatment was inoculated into five potted plants. After seven days, all the inoculated leaves displayed symptoms similar to those observed in the fields, whereas the control leaves did not exhibit these symptoms. The pathogenicity test was repeated three times. Following Koch's postulates, the pathogen was re-isolated and identified as C. cassiicola through morphological and ITS sequence each time. To further identify, we selected a representative isolate, LD-TJ, for multi-locus sequence analysis of its ITS, LSU, and TEF1-α genes (GenBank Accession Nos. PQ042036, PQ035023, PQ060235)(Voglmayr et al. 2017). BLASTN analysis of the sequences obtained showed a high similarity of 99 to 100% with the ITS (JAEMHE010000031.1:51214-51777, 481/481 nucleotides), LSU (JAEMHE010000018.1:728464-729373, 910/910 nucleotides), and TEF1-α (JAEMHE010000005.1:656736-657712, 969/970 nucleotides) sequences of C. cassiicola CC01. The phylogenetic tree showed that the LD-TJ strain, C. cassiicola CC01(isolated from rubber trees), and C. cassiicola CC_29(isolated from soybean leaves) clustered into a clade with a 99% bootstrap value. C. cassiicola was identified as the cause of patchouli leaf spot in Hainan(Chen et al. 2010), but it has not yet been reported in Guangdong. Identifying P. cablin leaf spot disease is crucial in Guangdong Province because it is the main growing area for P. cablin in China (Yan et al. 2021).

Protein P5 of pear chlorotic leaf spot-associated virus is a pathogenic factor that suppresses RNA silencing and enhances virus movement.

Pear chlorotic leaf spot-associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single-stranded, negative-sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens-mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self-interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell-to-cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N-terminal 1-89 amino acids of the P5 were responsible for the self-interaction ability and RSS activity, for which the signal peptide at positions 1-19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement.

First Report of Bacterial Leaf Spot Disease on Ginger Caused by Enterobacter quasiroggenkampii in China.

In autumn 2023, an unknown leaf spot disease has occurred on ginger (Zingiber officinale Roscoe) in two fields of approximately 1800 m2 in Yongning District (22°49'N; 108°48'E), Nanning, China, with a incidence of 20-30%. The symptoms began as yellow spots on the leaves, expanding into elliptical to irregular lesions with yellow edges, the middle of the lesion turning grey-white in dry weather. Finally, multiple spots caused necrosis of the whole leaf. Twelve diseased leaves from six plants of two fields were collected, surface disinfected and ground. The ground samples were diluted and plated on nutrient agar (NA) medium at 28 °C for 48-72 h. The purified colonies appeared milky white and round, with smooth edges. Three isolates (GL1, GL2 and GL3) were selected for identification and pathogenic determination. They were gram negative, could utilize sorbitol, mannitol, inositol, raffinose, melibiose, disaccharides, and citrate; negative for methyl red, phenylalanine decarboxylase, hydrogen sulfide, urease; positive for voges-proskauer test and ornithine decarboxylase. These characteristics were consistent with Enterobacter genus (Wu et al., 2020). Genomic DNA was extracted from three isolates. The 16S rDNA region was amplified using 27F/1492R primers (Weisburg et al. 1991) and sequenced (accession no. PP837703-PP837705). Blastn analysis revealed that 16S rDNA sequences for GL1 was 99% identical (1373/1387 nt), GL2 96% (1364/1422 nt) and GL3 95% (1365/1435 nt) to Enterobacter quasiroggenkampii WCHECL1060 (NR_179166). To determine the species, the sequences of gyrB, rpoB and atpD genes were amplified using primers gyrB 01-F/gyrB 02-R, rpoB CM7/rpoB CM31b, and atpD 01-F/atpD 02-R, respectively (Lin et al. 2015; Zhu at al. 2010; Zhang et al. 2013). The GenBank accession numbers for the sequences were PP857680-PP857688. A multilocus phylogenetic tree was constructed with the concatenated sequence of 16S rDNA-gyrB-rpoB-atpD by using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA6 software. The three isolates clustered with E. quasiroggenkampii. Fifteen Darou ginger variety plants at the 4-5 leaf stage were tested for pathogenicity. Two to three leaves of each ginger plant were pricked with a syringe needle of 0.36mm in diameter or not and inoculated by spraying the bacterial suspension (108 CFU/mL), sterile water was used as a control. Five plants were inoculated with each isolate and the test was repeated three times. After 3-4 days of inoculation, all wounded leaves and about 10% of the unwounded leaves showed symptoms similar to those observed in the field. Control plants did not develop symptoms. Enterobacter quasiroggenkampii isolates were re-isolated from the inoculated leaves with symptoms, and their identity was confirmed by gyrB sequencing and colony morphology, completing Koch's postulates. Enterobacter quasiroggenkampii is a pathogen of humans that can cause nosocomial infections (Wu et al., 2020). In Guangxi, E. quasiroggenkampii was identified as one of the pathogens causing mulberry wilt (Jiao, 2022). To our knowledge, this is the first report of E. quasiroggenkampii causing bacterial leaf spot disease of ginger. The results of this study not only have practical significance for the control of ginger leaf spot, but also can provide excellent materials for the study of the differentiation and pathogenic mechanism of the genus Enterobacter, which has important academic value.

Results of the Mycological Investigation of the Shoot System of "Irinovsky Oak" Tree (Leningrad Oblast, Russia).

The wild nature monument "184-year-old oak tree in the village of Irinovka" (Leningrad oblast, Russia), better known in local historical literature as "Irinovsky Oak," was officially opened in 2013. It is represented by a separate pedunculate oak tree (Quercus robur), planted in 1829 and preserved in satisfactory condition at 194 years of age. This paper presents data from a survey of the shoot system of the tree investigated. A total of 12 fungal species were registered (Cladosporium herbarum, Colpoma quercinum, Coryneum depressum, Diatrypella quercina, Erysiphe alphitoides, Hyphoderma setigerum, Laetiporus sulphureus, Mar-chandiomyces corallinus, Peniophora quercina, Trichoderma viride, Sphaerulina quercicola, Vuilleminia comedens). The lichenicolous species Marchandiomyces corallinus was reported as a new find to the Leningrad oblast. It was shown that the shoot system of the tree investigated is associated with a complex of Quercus robur-adapted and coadapted species, among which such necrotrophs as Sphaerulina quercicola and Coryneum depressum and such pathogenic saprotrophs as Vuilleminia comedens and Colpoma quercinum, as well as saprotrophs (Diatrypella quercina, Peniophora quercina), dominated. This species complex ensures a continuous process of the crown's thinning.

Genome-wide analysis of the SWEET gene family and its response to powdery mildew and leaf spot infection in the common oat (Avena sativa L.).

The nutritional quality and yield of oats (Avena sativa) are often compromised by plant diseases such as red leaf, powdery mildew, and leaf spot. Sugars Will Eventually be Exported Transporters (SWEETs) are newly identified sugar transporters involved in regulating plant growth and stress responses. However, the roles of SWEET genes in biotic stress responses remain uncharacterized in oats. In this study, 13 AsSWEET genes were identified across nine chromosomes of the oat genome, all of which were predicted to contain seven transmembrane regions. Phylogenetic analysis revealed four clades of AsSWEET proteins, with high homology to SWEET proteins in the Poaceae family. Collinearity analysis demonstrated strong relationships between oat and Zea mays SWEETs. Using subcellular localization prediction tools, AsSWEET proteins were predicted to localize to the plasma membrane. Promoter analysis revealed cis-acting elements associated with light response, growth, and stress regulation. Six AsSWEET proteins were predicted to interact in a network centered on AsSWEET1a and AsSWEET11. Gene expression analysis of two oat varieties, 'ForagePlus' and 'Molasses', indicated significant expression differences in several AsSWEET genes following infection with powdery mildew or leaf spot, including AsSWEET1a, AsSWEET1b, AsSWEET2b, AsSWEET3a, AsSWEET11, and AsSWEET16. These SWEET genes are potential candidates for disease resistance in oats. This study provides a foundation for understanding the regulatory mechanisms of AsSWEET genes, particularly in response to powdery mildew and leaf spot, and offers insights for enhancing oat molecular breeding.

First report of leaf spot on Gleditsia sinensis caused by Colletotrichum gloeosporioides in China.

Gleditsia sinensis Lam (Lamarck et al., 1788) is an endemic species widely distributed in China. In Sep. 2022, leaf spot symptoms were observed on G. sinensis in Xuhui district (31◦9'16''N, 121◦26'36''E), Shanghai, China, with an incidence rate of 55% in the examination of 9 trees. The leaves showed typical symptoms of anthracnose with irregular gray-brown spots and sunken areas. For isolation, 5 × 5 mm sections were cut from the lesion edge of 20 infected leaves collected from 2 trees. The surface of the sections was sterilized by immersion in 75% ethanol for 30 s, followed by 5% NaClO for 1 min, rinsed three times with sterile water, and dried on sterile filter paper. These sections were placed on PDA plates incubated at 25°C in darkness. Eighteen isolates with similar colony morphology were obtained and purified by single spore culturing. Two isolates (YKY2301, 2302) from separate trees were further tested. On the 6th day, the colonies had a diameter of 7.6 to 8.4 cm and appeared white to gray-white with aerial hyphae. The colony's central part exhibited an orange hue due to the conidia accumulation, while the undersides displayed an orange-yellow color. The hyphae were hyaline and smooth, with septa and branches, and the conidia were cylindrical with blunt to slightly rounded ends, measuring 13.1 to 18.8 (average 15.9) µm× 4.0 to 6.6 (average 5.4) µm (n=184). From conidia germinated on glass slides, the appressoria measured 5.5 to 6.3 µm ×4.9 to 5.1 µm (n=50) and were nearly spherical or elliptical in shape. These characteristics matched those of the Colletotrichum gloeosporioides species complex (Cannon et al., 2012; Weir et al., 2012). For molecular identification, the genomic DNA was extracted using a modified CTAB method (Luo et al., 2012). Gene fragments including ITS (PP125667, PP125668), GAPDH (PP153428, PP153429), ACT (PP153424, PP153425), TUB2(PP153917, PP190256), and ApMAT (PP153426, PP153427) were obtained by PCR using universal primers (Huang et al., 2022) and sequenced. The sequences exhibited 98.19% to 99.82% identity with the corresponding gene of the type strain C. gloeosporioides IMI356878 (JX010152, JX010056, JX009531, JX010445, JQ807843) in NCBI BLAST. A multilocus Maximum likelihood phylogenetic tree was constructed based on concatenated the five genes by PhyloSuite. It showed that YKY2301, 2302 were on the same branch with C. gloeosporioides. Based on these results, the isolates were identified as C. gloeosporioides. Pathogenicity tests were conducted by mycelial and conidia inoculation. 5 mm mycelial or blank agar plugs were inoculated onto the leaves of 2 healthy trees in a garden (25 to 30 °C), with and without wounds made by toothpick pricking (n≥3 per group). All mycelial inoculated leaves showed leaf spots on the 6th day. Three healthy 2-year-old seedlings were inoculated with either conidia (108 conidia/ml) or water by leaf spray, and maintained in a climate chamber (27 °C, 80% humidity). Inoculated seedlings showed necrotic leaf spots on day 14, and wilted within 3 weeks. The controls in all tests remained asymptomatic. The pathogen has been re-isolated and confirmed by sequencing, thus fulfilling Koch's postulates. This is the first report of leaf spots caused by C. gloeosporioides on G. sinensis in the world. As illustrated by the example of legume pod infection (Gerusa et al., 2019), it poses a potential threat to the fruits of G. sinensis, despite currently only affecting their ornamental value. This report provides basic information for future research.

Effect of fumigants on inoculum of Neopestalotiopsis spp. in strawberry crowns and soil.

Florida's strawberry industry has been facing an emerging threat after several outbreaks of an aggressive Neopestalotiopsis sp. affecting fruit, leaf, and crown caused severe yield losses. Our studies found Neopestalotiopsis sp. can survive from one production season to the next on crop debris in soil, which led us to evaluate the effect of fumigants on resident inoculum in strawberry crowns and soil. Different rates of 1,3-dichloropropene, chloropicrin, and metam potassium were applied to pasteurized soil inside glass jars containing infected strawberry crowns. Soil samples were taken after broadcast and bed fumigation treatments with a combination of 1,3-dichloropropene/chloropicrin at ratios of 63:35 were applied at four commercial fields. Crowns and soil samples were processed and plated on a semi-selective medium for Neopestalotiopsis spp. and colony-forming units (CFU) were counted 5 days after plating. CFU counts in crowns treated with 1,3-dichloropropene, chloropicrin, and metam potassium decreased significantly as rates increased and were described by exponential decay models. CFUs were not recovered in most of the soil samples from fumigated strawberry beds after broadcast or bed fumigation. However, CFUs were found in non-fumigated row middles between fumigated beds which can serve as a source of inoculum to start new epidemics. Chloropicrin, 1,3-dichloropropene, and metam potassium were effective on reducing Neopestalotiopsis spp. inoculum in strawberry crowns and soil, providing new evidence on the fungicidal activity of 1,3-dichloropropene. Broadcast fumigation with 1,3-dichloropropene/chloropicrin at ratios of 63:35 could potentially be used to reduce inoculum of Neopestalotiopsis sp. in severely infested fields.

Biological characterization and in vitro fungicide screening of a new causal agent of walnut leaf spot in Guizhou Province, China.

Walnut (Juglans regia L.) is a widely grown nut plant worldwide, including in Guizhou Province, located in southwest China. The high quality and special taste make Guizhou walnuts, particularly those produced in Hezhang County, a "Chinese National Geographical Indication Product" that substantially contributes to the local economy and grower's income. In July 2022, a serious occurrence of leaf spot disease was observed in a walnut plantation area, Shuitang Town, Hezhang County, Guizhou Province, China (27°07'67″N, 104°64'61″E). The causal agent was identified as Didymella segeticola through morphological characterization and amplification and sequencing of the internal transcribed spacer (ITS) region, beta-tubulin (TUB) gene, and glyceraldehyde-3-phosphate dehydrogenase (G3PD) gene. Koch's postulates, including re-isolation and identification, were performed to confirm its pathogenicity on healthy leaves. To our knowledge, this is the first report of D. segeticola causing leaf spot on walnuts worldwide. Further, to determine its biological characteristics, which could be utilized for future disease management, the effects of temperature, light, and carbon and nitrogen resources on mycelial growth, conidia production, and conidia germination and the effects of humidity on conidia germination were studied. The optimum temperature for mycelial growth of representative strain D. segeticola C27 was 20°C. Increasing the light period significantly decreased conidia production and conidia germination. Maltose and beef extract were the best carbon and nitrogen sources, respectively, for the pathogen. Conidia germination was enhanced at 90% humidity. In vitro screening of effective fungicides was conducted. Among the 20 screened fungicides, difenoconazole showed the best inhibition rate, with an EC50 (concentration for 50% of the maximal effect) of 0.0007 µg/mL. Tetramycin also showed sufficient inhibitory effects against D. segeticola, with an EC50 value of 0.0009 µg/mL. Our study provides new insights into the causal agent of walnut leaf spot in Guizhou, China, as well as the first pathogen characteristics and promising candidate fungicides for its management.

First Report of Alternaria alternata Causing Alternaria Leaf Spot of Black Nightshade in China.

Black nightshade (Solanum nigrum L.) belongs to Solanaceae family, which is distributed all over China and has become one of the malignant weeds in cotton field in Xinjiang (Zhang et al., 2020; Zhang et al., 2021). During July 2016 to August 2023, leaf spot was observed on black nightshade in Hami and Shihezi city, Xinjiang, China. The spots appeared as brown circular or irregular, with some small black dots surrounded with yellow halos. Twenty five black nightshade plants were collected from a cotton field (100 m2) for pathogen isolation in Shihezi, July 2023. Leaf samples (2×2 mm) from the boundary between diseased tissue and healthy tissue, sterilized in 70% ethanol for 45 s, dipped in 2% NaClO for 30 s, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) after dried and cultivated at 25°C in the dark for 4 days. Twenty-five fungal isolates were obtained and purified by using the single-spore isolation method, and all isolates were found to be similar in morphology, appeared as dark brown and produced dark brown pigmentation on PDA. Conidiophores were black brown, 8 to 58 × 10 to 20 µm, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, the results similar to those of Alternaria alternata isolated from buckwheat (Li et al., 2021). To confirm this identification, total genomic DNA of the isolates were extracted by CTAB method (Doyle et al., 1987). The internal transcribed spacer (rDNA-ITS), Alternaria major allergen gene (Alt α1) and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) of the A. alternata 26 were amplified using the primers ITS1/ITS4 (Glass et al., 1995), Altα1-F/Altα1-R (Li et al., 2024) and gpd-F/gpd-R (5'-CAACGGCTTCGGTCGCAT- TG-3' / 5'-GCCAAGCAGTTGGTTGTG-3'), respectively. The rDNA-ITS, Altα1 and GAPDH gene sequences were deposited in GenBank (KX904867, PP263361, PP263360), and showed 100% identity (rDNA-ITS: 572 out of 609 bp; Alt α1: 514 out of 516 bp; GAPDH: 619 out of 619 bp) to A. alternata (KU179665; MW522975; MK451977), respectively. The multi-gene phylogenetic tree showed that the representative isolate was grouped with A. alternata and identified as A. alternata. Black nightshade was used for pathogenicity assay. Fifteen plants at 3 to 4-leaf stage were selected and sprayed with the conidial suspension (1.0×106 spores/mL) of A. alternata 26 after wound the underside of leaves (2 mm length) with inoculating needle. Five plants were sprayed with sterilized water as control. After inoculation, placed the plants in a plastic box and covered with plastic wrap to keep 80% relative humidity for 3 days at 25℃ in the greenhouse, then removed the plastic box. Symptoms of leaf spots appeared within 12 - 15 days that were similar to the symptoms observed in the field, and the fungal pathogen that was re-isolated from symptomatic leaves was identical to original pathogen on the basis of morphological and molecular analysis to fulfill Koch's postulates. This experiment was repeated three times, and the disease incidence was above 80% after inoculation at each time. To our knowledge, this is the first report of A. alternata causing leaf spot of black nightshade in China. This report will help us to monitoring distribution of the disease and providing theoretical basis for disease prevention and control.

Investigating the Role of Viruses in the Rapid Decline of Young Apple Trees in High-Density Orchards in New York.

A sudden, unexplained decline and collapse of young apple trees on dwarfing and semi-dwarfing rootstocks has been reported across North America over the past decade. Although viruses have been detected in declining trees, no information is available on their potential causal role in the decline phenomenon. To this end, virus-inoculated apple trees were established in a high-density experimental orchard and monitored over five years. Tree decline was observed in year 4 (2022), resulting in 17% mortality, with declining trees exhibiting marked vascular tissue necrosis. However, none of the eight viruses and one viroid detected in the experimental orchard was significantly more prevalent in declining trees. Extreme temperature fluctuations in January 2022, followed by a severe water deficit in summer 2022, were recorded at the experimental orchard. Similar but distinct observations were made in a nearby commercial orchard with foliar nutrient imbalances documented in trees exhibiting symptoms of rapid decline. Together, our findings suggest that viruses are not primarily responsible for the rapid decline phenomenon and highlight the need for future work to investigate the roles of tree physiology and water stress in tree decline, as well as the potential efficacy of horticultural mitigation practices.

Identification of Cercospora spp. on corn in North America and baseline flutriafol fungicide sensitivity.

Gray leaf spot (GLS) is an important corn disease reportedly caused by Cercospora zeae-maydis and C. zeina. Recently, flutriafol, a demethylation inhibitor (azole) fungicide received EPA registration as Xyway® LFR®, a product that is applied at planting for management of fungal diseases in corn, including suppression of GLS. In this study, 448 Cercospora spp. isolates were collected in 2020 and 2021 from symptomatic corn leaf samples submitted from the United States and Ontario, Canada. The Cercospora spp. were identified using multi-locus genotyping of the internal transcribe spacer (ITS), elongation factor 1-α (EF1), calmodulin (CAL), histone H3 (HIS), and actin (ACT) gene. Based on the multi-locus phylogenetic analyses, six species were identified; C. cf. flagellaris (n = 77), C. kikuchii (n = 4), C. zeae-maydis (n = 361), Cercospora sp. M (n = 2), Cercospora sp. Q (n = 1), and Cercospora sp. T (n = 3). In subsequent pathogenicity tests using selected isolates from each of these species, only C. zeae-maydis resulted in symptoms on corn with no disease symptoms observed after inoculation with C. cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T. While disease symptoms were observed on soybean following inoculation with C. cf. flagellaris, C. kikuchii, and Cercospora sp. Q, but not the other three species. Fungicide sensitivity of Cercospora spp. to flutriafol was assessed using a subset of 340 isolates. The minimum inhibitory concentration (MIC) to inhibit the growth of Cercospora spp. completely was determined based on growth of each species on flutriafol-amended clarified V8 agar at nine concentrations. The EC50 was also calculated from the same trial by measuring relative growth as compared to the non-amended control. Cercospora zeae-maydis was sensitive to flutriafol with mean MIC values of 2.5 µg/mL and EC50 values ranging from 0.016 to 1.020 µg/mL with a mean of 0.346 µg/mL. Cercospora cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T had mean EC50 values of 1.25 µg/mL, 7.14 µg/mL, 2.48 µg/mL, 1.81 µg/mL, and 2.24 µg/mL respectively. These findings will assist in monitoring the sensitivity to the flutriafol fungicide in Cercospora spp. populations.

Colletotrichum Species Associated with Apple Bitter Rot and Glomerella Leaf Spot: A Comprehensive Overview.

Species of the genus Colletotrichum are among the most important plant pathogens globally, as they are capable of infecting many hosts-apple (Malus spp.) and other fruit and woody plant species-but also vegetable crops, cereals, legumes, and other annual and perennial herbaceous plants. The apple (Malus spp.) is attacked by various species from the genus Colletotrichum, whereby 27 different species from this genus have been described as the causative agents of apple bitter rot (ABR) and 15 as the cause of Glomerella leaf spot (GLS). These species generally belong to one of three species complexes: Colletotrichum acutatum, Colletotrichum gloeosporioides, and Colletotrichum boninense. The largest number of apple pathogens of the genus Colletotrichum belong to the species complex C. acutatum and C. gloeosporioides. However, further data on these species and the interactions between the species complexes of the genus Colletotrichum that cause these two apple diseases is needed for the development of effective control measures, thus ensuring successful and profitable apple cultivation. To contribute to this endeavor, a comprehensive review of the causative agents of ABR and GLS from the genus Colletotrichum is provided. In addition to presenting the species' current names, distribution, economic significance, and the symptoms they cause in apple, their development cycle, epidemiology, and molecular detection strategies are described, with a particular emphasis on control measures.

The occurrence and mechanism of field resistance to boscalid and pyraclostrobin in Stemphylium solani, the causal agent of tomato gray leaf spot in China.

The destructive disease gray leaf spot, caused by Stemphylium solani, is prevalent in tomato plants in China. A variety of fungicides have been extensively used for controlling the disease, with a particular focus on succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, there was a lack of information regarding the resistance of S. solani to boscalid (SDHI) and pyraclostrobin (QoI) in China. In this study, the sensitivity of S. solani to boscalid and pyraclostrobin was monitored. The EC50 values for boscalid ranged from 0.02 to 3.0 µg∙mL-1, with an average value of 0.62 µg∙mL-1, while the EC50 values for pyraclostrobin ranged from 0.21 to 14.71 µg∙mL-1, with an average value of 6.03 µg∙mL-1. Based on these findings, the frequencies of observed resistance were as follows: 36.7% for boscalid and 50% for pyraclostrobin; while the resistance frequency to both boscalid and pyraclostrobin in S. solani was 19.4%. The mutation associated with boscalid resistance in S. solani within tomato fields was identified as SdhB-H277Y, while the mutation related to pyraclostrobin resistance was found in cytochrome b, specifically Cytb-G143A. The resistant mutants displayed diminished fitness in terms of mycelial growth, yet their pathogenicity exhibited no significant disparities. To delay the development of resistance, it is advisable to employ a rotation strategy using alternative fungicides with different modes of action or mix with fungicides with multi-site-contact activity for disease management.

Enhancing Resistance to Cercospora Leaf Spot in Mung Bean (Vigna radiata L.) through Bradyrhizobium sp. DOA9 Priming: Molecular Insights and Bio-Priming Potential.

Mung bean (Vigna radiata L.), a vital legume in Asia with significant nutritional benefits, is highly susceptible to Cercospora leaf spot (CLS) caused by Cercospora canescens, leading to significant yield losses. As an alternative to chemical fungicides, bio-priming with rhizobacteria can enhance plant resistance. This study explores the potential of Bradyrhizobium sp. strain DOA9 to augment resistance in mung bean against CLS via root priming. The results reveal that short (3 days) and double (17 and 3 days) priming with DOA9 before fungal infection considerably reduces lesion size on infected leaves by activating defense-related genes, including Pti1, Pti6, EDS1, NDR1, PR-1, PR-2, Prx, and CHS, or by suppressing the inhibition of PR-5 and enhancing peroxidase (POD) activity in leaves. Interestingly, the Type 3 secretion system (T3SS) of DOA9 may play a role in establishing resistance in V. radiata CN72. These findings suggest that DOA9 primes V. radiata CN72's defense mechanisms, offering an effective bio-priming strategy to alleviate CLS. Hence, our insights propose the potential use of DOA9 as a bio-priming agent to manage CLS in V. radiata CN72, providing a sustainable alternative to chemical fungicide applications.

First Report of Leaf Spot Caused by Didymella segeticola on Rosa roxburghii Tratt in China.

Rosa roxburghii Tratt, known as Cili in China, is a fruit crop that grows in the mountains of southwest China at altitudes of 500 - 2500 m, especially in Guizhou province (Huang et al. 2022). In July 2021, leaf spot symptoms were observed on approximately 20 to 30% of R. roxburghii plants in a field of 6,000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou Province, China. Severe leaf spot can lead to excessive leaf drop, significantly weakening the tree and adversely affecting its growth and fruit quality, which in turn can result in reduced or even lost harvests. The symptoms appeared as irregular brown spots (0.5 to 9.5 mm), which could coalesce when densely clustered and could lead to yellowing of the leaves in severe cases. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees. Symptomatic leaves were washed with sterile distilled water and then portions of the tissue (0.5×0.5cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol, 2 min with 3% NaOCl, washed three times with sterilized distilled water), the leaves were dried and placed flat on potato dextrose agar (PDA) and left for 3-4 days incubated at 25°C (Fang, 2007). From this process, three isolates, denoted as F3-Y-21, F3-Y-22 and F3-Y-23, were obtained through single spore isolation, all displaying identical morphology. Subsequently, isolate F3-Y-21 was selected for further study. The colonies had dense aerial hyphae, initially white and later turning gray near the colony center when cultured on PDA at 28℃. Pycnidia were dark, spherical or flat spherical, and 42.2 to 52.6 µm × 51.5 to 55.2 µm in diameter (n = 50). Conidia were oval, smooth, aseptate, usually guttulate, and the size was 3.0 to 4.6 µm × 2.3 to 2.8 µm (n = 50). These morphological attributes were consistent with the description of Didymella segeticola (Chen et al. 2015). The isolate F3-Y-21 was confirmed to be D. segeticola by amplification and sequencing of the rDNA internal transcribed spacer region (ITS; primers ITS5/ITS4), large subunit ribosomal RNA gene (LSU; primers LROR/LR5), beta-tubulin gene (TUB2; primers Bt2a/Bt2b), and RNA polymerase II second largest subunit gene (RPB2; primers RPB2-5F2/fRPB2-7cR) (Liu et al. 1999; Suwannarachetal. 2019). Sequences from PCR amplification were deposited in GenBank under accessions PP159078 (ITS), PP159081 (LSU), PP178656 (TUB2), and PP178653 (RPB2). BLASTn searches of the sequences in GenBank revealed 100.00% identity of ITS (486/486 bp), 100.00% identity of LSU (574/574 bp), 98.93% identity of TUB2 (277/280 bp), and 99.05% identity of RPB2 (838/846 bp) with those sequences of D. segeticola CGMCC 3.17489 (accessions KP330443, KP330455, KP330399, and KP330414, respectively). A phylogenetic tree was constructed by MEGA7.0 using the maximum likelihood method. The isolate F3-Y-21 clustered in the same branch with D. segeticola. To assess its pathogenicity, a pot assay was conducted. Twelve leaves of three healthy R. roxburghii plants were spray-inoculated with a spore suspension (106 spores/ml), and an additional three plants were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The experiment was repeated three times. After 7 days, the inoculated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The pathogen was reisolated from diseased leaves and identified by morphological characterization and molecular analyses (ITS, LSU, TUB2 and RPB2), and the reisolated pathogen was identical to D. segeticola, thus fulfilling Koch's postulates. Similar results were obtained from three replications of the pathogenicity test. To our knowledge, this is the first report of leaf spot diseases of R. roxburghii plants caused by D. segeticola in China, although it has been previously reported to cause diseases on other hosts in China (Guo et al. 2020). It provides a theoretical basis for the detection and prevention of R. roxburghii leaf spot disease.

First report of Aspergillus tubingensis causing leaf spot on Pogostemon cablin in China.

Patchouli (Pogostemon cablin (Blanco) Benth) is an important medicinal and aromatic plant widely cultivated in China, India, and other Southeast Asian countries. It is renowned for its diverse applications in traditional medicine and its detoxification, antibacterial, anti-inflammatory, and other pharmacological properties (Wu et al. 2016; Fang et al. 2022). In May 2023, a severe leaf spot disease was observed on Pogostemon cablin plants grown in most plantations in Yulin, Guangxi, China (22°26'N; 109°83'E), with over 50% incidence rate. Symptoms began as small, circular, brown spots on leaves, enlarging with yellow halos. Lesions expanded into irregular shapes with necrotic centers. Advanced stages showed extensive yellowing, browning, and leaf senescence. A total of 20 symptomatic plants were sampled from 5 different locations within the detected area, with 4 plants sampled per location. To isolate the pathogen, 20 affected leaves were collected from these plants and preliminarily washed with sterile distilled water (SDW). Five small tissue pieces (5×5 mm) were excised from the lesion edge of each leaf, surface-disinfected with 75% ethanol and 1% NaClO, rinsed thrice with SDW, and placed on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Out of these, 18 plants (90%) yield fungal isolate with recurrent and similar morphological characteristics. Four representative isolates (X5-1-1, X5-1-3, X5-1-5, and X5-1-7) were selected for further analysis. On PDA, colonies were initially white, gradually turning black on the surface, with light yellow on the reverse side of the plate. Conidia were brown to black, globose, rough-walled, and 2.6 to 5.2 µm in diameter. Conidial heads were brown-black, and conidiophores were smooth and hyaline. Morphological characteristics matched those of Aspergillus sp. (Guo et al. 2017). For molecular identification, the internal transcribed spacer (ITS) region and the ß-tubulin (TUB) gene of all four isolates were sequenced (Lim et al. 2019). All four isolates (X5-1-1, X5-1-3, X5-1-5, and X5-1-7) showed consistent morphological characteristics and 100% identical ITS and TUB sequences. Representative sequences from isolate X5-1-5 were submitted to GenBank (ITS: PP789632; TUB: PP798205). The obtained ITS and TUB sequences showed 99% similarity to Aspergillus tubingensis (ITS: OP737633; TUB: MG991377). Based on morphological and molecular analyses, the fungus was identified as A. tubingensis (Palmer et al. 2019). For pathogenicity tests, a spore suspension (1 × 10^6 conidia/mL) was prepared from 7-day-old cultures of A. tubingensis grown on PDA. The suspension was sprayed onto leaves of 10 healthy Pogostemon cablin plants until runoff. Control plants were sprayed with SDW. All plants were kept in a controlled greenhouse (12/12h light/dark, 25 ± 2 °C, 90% humidity). After 7 d, symptoms identical to those observed in the field developed on all pathogen inoculated plants, while control plants remained asymptomatic. The fungus was successfully re-isolated from infected leaves in three successive trials, fulfilling Koch's postulates. Notably, A. tubingensis has previously been reported causing field diseases on strawberry in California, Jatropha curcas and Helleborus species in China (Palmer et al. 2019; Guo et al. 2017, Liaquat et al. 2019), and vine canker on table grape in Italy (Vitale et al. 2012). To our knowledge, this is the first report of A. tubingensis causing leaf spot on Pogostemon cablin in China. This finding provides a foundation for further investigate into the biology, epidemiology, and management of this disease.

First Report of Cercospora chevalieri Causing A Leaf Spot Disease on Amorphophallus konjac in China.

Amorphophallus konjac, commonly called voodoo lily, is a cash crop widely cultivated in southwest China (Gao et al. 2022). In August 2022, leaf spot symptoms were observed in a field (1 ha) located at Fuyuan (25.67°N; 104.25°E), Yunnan, China, resulting in substantial economic losses. Brown lesions, with an incidence ranging from 20 to 40%, typically had a whitish or gray center and were surrounded by yellow halos. Microscopic observations of the spots revealed anamorphic species Cercospora chevalieri. Conidiophores were 50-150 × 4-7 µm, cylindrical, unbranched, smooth-walled, pale brown and aggregated in dense fascicles arising from a brown stroma. The conidiogenous cells were integrated, terminal or intercalary, pale brown to brown and proliferated sympodially. The conidiogenous loci were thickened and darkened, and 2-3 µm in diam. The conidia were formed singly, obclavate-cylindrical, 90-160 × 5-7 µm, with an average of 130 × 6 µm (n = 30), 6-11 septa, thin-walled, smooth, hyaline or subhyaline, straight or curved with an obtuse apex and obconically truncate base, with thickened and darkened hilum. These morphological characteristics matched those of C. chevalieri, the causal agent of leaf spot on A. paeoniifolius (Braun et al. 2014; Saccardo et al. 1913). A conidial suspension in sterile water from lesions was used to inoculate water agar, and germinated conidia were transferred to potato dextrose agar（PDA) and incubated at 27°C for 7 days. Induction of sporulation was unsuccessful using PDA, as well as malt extract agar, potato sucrose agar and synthetic nutrient-poor agar. Two out of ten isolates were selected for molecular identification and pathogenicity assay. Genomic DNA from two pure isolates (KUNCC22-12536 and KUNCC22-12537) was extracted for PCR and amplified with primers for the internal transcribed spacers (ITS: ITS1/ITS4), calmodulin (CMD: CAL228F/CAL2Rd), translation elongation factor 1-alpha (TEF1-α: 728F/986R), actin (ACT: 512F/783R), histone H3 (HIS3: CYLH3F/CYLH3R), beta-tubulin gene (TUB2: BT-1F/BT-1R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Gpd1/Gpd2), respectively (Vaghefi et al. 2021). The newly generated sequences for ITS (OP719153/OP719154), CMD(OP740904/OP740905), TEF1-α (OP740910/OP740911), ACT (OP740902/OP740903), HIS3 (OP740908/OP740909), TUB2 (OP740912/OP740913), GAPDH (OP740906/OP740907) of C. chevalieri were submitted to GenBank. So far, no sequence data of C. chevalieri were available in the GenBank database. As expected, most genes (TEF1-α, ACT, CMD, HIS, TUB2 and GAPDH) showed 91 to 95% identity to their best hits within species of the genus Cercospora. The phylogenetic tree showed that sequences retrieved from two isolates obtained from the A. konjac leaf spots clustered together within Cercospora forming a strongly supported clade. To test Koch's postulates, ten four-month-old healthy A. konjac plants grown in pots were used for a pathogenicity test in a greenhouse. One leaf of each plant was inoculated with mycelial plugs, and one leaf was inoculated with a sterile PDA plug. These plants were enclosed in plastic bags for 72 h. Only leaves inoculated with mycelium plugs produced brown lesions, which appeared after 10 to 14 days on inoculated leaves. Control plants treated with sterile PDA plugs remained asymptomatic. This experiment was repeated twice with the same results. C. chevalieri was reisolated from infected leaves and identified based on morphology and Sanger sequencing of the ITS region. To our knowledge, this is the first report of C. chevalieri causing leaf spot on A. konjac and the first report of this species from China (Braun et al. 2014), which provides key information for diagnosis and management of this disease.

Septoria cannabicola, a new species on Cannabis sativa from Japan.

Septoria leaf spot on hemp has re-emerged with increasing hemp cultivation worldwide. In Japan, Septoria cannabis , initially recorded as the causal pathogen in Japan, was studied with morphology based on the current criteria and detailed molecular phylogenetic analyses using seven gene loci. The robust phylogenetic data and morphology of examined specimens unveiled the existence of a new species of the genus Septoria causing leaf spot disease on Cannabis sativa .

Rapid Identification of Phytotoxins Produced by Glomerella cingulata Using High-Resolution Mass Spectrometry-Based Qualification, Targeted Structural Confirmation and Their Characteristics Investigation.

Glomerella cingulata is a pathogenic fungus that can cause apple Glomerella leaf spot (GLS), a new and destructive apple disease in China. Phytotoxins are important factors closely related to the disease process, but there is still no report on the phytotoxins of G. cingulata. The aim of this study was to rapidly identify the phytotoxins of this pathogen using a strategy of HRMS-based preliminary qualification, followed by targeted structure confirmation and also investigation of phytotoxicity characteristics. First, the crude toxin sample was directly analyzed by the UPLC-HRMS and GC-MS, and the data were processed to screen for possible phytotoxic compounds using MS library and the phytotoxicity-related literature. The reference standards of credible phytotoxic compounds were then subjected to targeted structure validation (signal comparison between standards and compounds in crude toxin via HPLC-DAD, UPLC-MS/MS, and GC-MS), and also the phytotoxicity assay. The results confirmed six phytotoxins produced by G. cingulata, namely 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 2,5-bis(hydroxymethyl)furan (BHMF), 2-furoic acid (FA), 2,3-butanediol, trans-aconitic acid (TAA), and cis-aconitic acid (CAA). Of these, HMFCA and TAA exhibited greater phytotoxicity. Main characteristics: All of them were non-host-selective toxins, and toxins were synergistically phytotoxic to the host when mixed. BHMF, HMFCA, FA, TAA, and CAA could be commonly produced by all tested strains, and their phytotoxicity can be significantly inhibited or even eliminated at high temperatures or high pH. The elucidation of the phytotoxins of G. cingulata in this work could provide information on the pathogenesis and control of apple GLS.