Brown Spot Research Articles

Search for sources of resistance to leaf diseases among present winter bread wheat varieties in the southern part of the Rostov region

The purpose of the current work was to search for sources of winter wheat varieties’ resistance to the most common pathogens of the area under artificial infectious backgrounds and recommend them for inclusion in the breeding process. The study was carried out on the experimental plots of the laboratory for plant immunity and protection of the FSBSI Agricultural Research Center “Donskoy” in 2021–2023. In order to expand the genetic diversity in immunity of newly developed varieties, it is necessary to use new sources of resistance. The objects of study were 78 winter wheat varieties from interstation variety testing, representing various breeding institutions in Russia and some foreign varieties. The study material was presented by the North Caucasian populations of pathogens of such wheat leaf diseases as powdery mildew, brown and yellow rust, and leaf spot (Septoria). Infectious backgrounds were formed according to generally accepted methods, using spore material of pathogens both stored and collected from crops in the previous year (types of rust) and collected from overwintered plants in the spring (powdery mildew, leaf spot). Weather conditions varied across the years of study (mainly in the autumn), but the damage to susceptible test varieties in the experiments was maximum. As a result of the study of the varieties on their resistance to one pathogen, there have been identified 23 varieties resistant to powdery mildew, 56 ones to leaf rust, 47 ones to yellow rust, 8 varieties to leaf spot. There has been given a characteristic of varieties according to various degree of resistance or susceptibility to each pathogen. On the resistance to two pathogens, there have been identified 17 varieties resistant to brown and yellow rust, 2 varieties resistant to brown rust and powdery mildew, 2 varieties resistant to yellow rust and powdery mildew, 1 variety resistant to brown rust and leaf spot. Ten varieties have shown resistance to no one pathogen, but they have had moderate resistance or moderate susceptibility to several other pathogens. 20 varieties were resistant to three diseases in various combinations, and 16 of them were resistant to brown, yellow rust, and powdery mildew. One variety ‘MV Nador’ from Hungary showed resistance to all 4 pathogens. All resistant varieties identified in the study can replenish the supply of winter wheat sources to the complex of leaf diseases for breeding purposes or be used in integrated crop protection against leaf diseases.

First report of Alternaria alternata causing leaf spot on Polygonatum kingianum in China.

Polygonatum kingianum is a Chinese herbal medicine that belongs to the genus Polygonatum of the family Liliaceae. In June 2023, Polygonatum kingianum Coll. et Hemsl. in nurseries in Qujing, Yunnan Province, China, showed irregular brown spots on the leaves, whole leaf necrosis, and plant death in serious cases, with an incidence of 10-20% (Fig. S1). To identify the pathogens of P. kingianum, six diseased samples were collected from nurseries with 0.6 acre. These diseased sample leaves were soaked in 0.1% HgCl2 for 1 min and 75% ethanol for 2 min and then rinsed thrice with sterile water. Treated leaves were cut into small pieces (5×5 mm) and cultured on potato dextrose agar (PDA) for five days at 28°C. Total thirteen fungal strains were isolated from PDA medium. The nuclear ribosomal internal transcribed spacer of ribosomal DNA (ITS rDNA) region of these 13 strains was amplified by polymerase chain reaction (PCR) using universal primers ITSI/ITS4 (White et al. 1990). Sequencing and BLAST of the ITS region on NCBI showed that 11 out of 13 fungal strains belonged to the genus Alternaria, with an identity ≥99%. We selected one of the Alternaria strains, HJ-A1, for further study. The HJ-A1 colony appeared grayish brown white-to-gray with a flocculent texture on the front side and a dark gray underside on the PDA medium (Fig. S1). The conidiophores appeared brown, either single or branched, and produced numerous short conidial chains. The conidia were obclavate to obpyriform or ellipsoid in shape and contained 1-4 transverse septa and 0-2 oblique septa. The conidial diameter was 27.30µm in length and 12.27µm in width. (Fig. S1). To further determine the species of HJA1, the genomic DNA of HJ-A1 was extracted using the Lysis Buffer for PCR (AG, Hunan, China). Four Alternaria genomic DNA regions including the ITS, translation elongation factor 1-α gene (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen gene (Alt a1) were amplified by PCR using the primers as previously reported (Woudenberg et al. 2013, Hong et al. 2005). Sequence analysis revealed that the ITS (484bp) of HJ-A1 (NCBI No. PP082633), TEF1-α (267bp) of HJ-A1 (NCBI No. PP419893), GAPDH (582bp) of HJ-A1 (NCBI No. PP419892), and Alt a1 (522bp) of HJ-A1 (NCBI No. PP228046) shared the highest identity with A. alternata respectively (99≥%). A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, GAPDH, TEF, and Alt a1 using MEGA 7. The results showed that HJ-A1 strain clustered with A. alternate (Fig. S2). The pathogenicity of HJ-A1 was tested according to Koch's postulates by inoculating HJ-A1 conidia suspension (2×105 conidia/mL) into leaves of 1-year-old P. kingianum, with sterile water as a control. Each treatment group included 3 plants with 3 replicates. The tested plants were planted in a phytotron at 28℃ and 90% humidity. Three days after inoculation, symptoms similar to those under natural conditions were observed in the HJ-A1-inoculated plants, whereas no symptoms were observed in the control plants (Fig. S1). The same fungal strains were re-isolated from inoculated leaves and identified by morphologically and sequence of ITS. Previous studies showed that Alternaria alternata funji cause many plant diseases, such as fig fruit rot (Latinović N et al. 2014)，daylily leaf spot (Huang D et al. 2022), fruit blight on sesame (Cheng H et al. 2021)，leaf spot of Cynanchum atratum Bunge (Sun H et al. 2021) and so on. To our knowledge, this is the first report of A. alternata causing P. kingianum leaf spot in China. The discovery of this pathogen will help to guide the protection and control of P. kingianum disease.

Identification of plant based potential antifungal compounds against BMK-1 protein of Bipolaris oryzae using molecular docking approach

Rice brown spot is an important disease of rice worldwide that inflicts substantial yield losses. The antimicrobial potential of methanol, acetone and dimethyl sulfoxide (DMSO) extracts of different medicinal plants, viz., Syzygium aromaticum, Saussurea costus, Acorus calamus, Bergenia ciliate, Geranium pratense, Mentha longifolia, Inula racemosa, Podophyllum hexandrum, Heracleum candicans and Picrorhiza kurroa, against the brown spot pathogen Bipolaris oryzae in vitro was evaluated via mycelial growth inhibition and spore germination inhibition assays. Among the plant extracts tested, 100% mycelial inhibition was observed for the methanol extract of Syzygium aromaticum at all three concentrations (2000 ppm, 3000 ppm and 4000 ppm), followed by the methanol extract of Inula racemosa (90.33%) at 4000 ppm. A maximum conidial germination inhibition of 83.54% was exhibited by the Heracleum candicans leaf extract. Phytochemical profiling of Syzygium aromaticum and Inula racemosa through liquid chromatography and mass spectrometry (HR-LCMS) revealed the presence of several compounds, such as eugenol, ursolic acid, quercetin, chlorogenic acid, and noscapine. A molecular docking approach was used to identify key inhibitory molecules against B. oryzae. Among the compounds detected in S. aromaticum and Inula racemosa, ursolic acid and noscapine were found to have the greatest binding affinity for the Big Mitogen Activated Protein Kinase (BMK-1) enzyme present in B. oryzae. In conclusion, S. aromaticum and Inula racemosa are potent compounds that could serve as lead compounds for drug discovery in the future.

Network analyses predict major regulators of resistance to early blight disease complex in tomato

BackgroundEarly blight and brown leaf spot are often cited as the most problematic pathogens of tomato in many agricultural regions. Their causal agents are Alternaria spp., a genus of Ascomycota containing numerous necrotrophic pathogens. Breeding programs have yielded quantitatively resistant commercial cultivars, but fungicide application remains necessary to mitigate the yield losses. A major hindrance to resistance breeding is the complexity of the genetic determinants of resistance and susceptibility. In the absence of sufficiently resistant germplasm, we sequenced the transcriptomes of Heinz 1706 tomatoes treated with strongly virulent and weakly virulent isolates of Alternaria spp. 3 h post infection. We expanded existing functional gene annotations in tomato and using network statistics, we analyzed the transcriptional modules associated with defense and susceptibility.ResultsThe induced responses are very distinct. The weakly virulent isolate induced a defense response of calcium-signaling, hormone responses, and transcription factors. These defense-associated processes were found in a single transcriptional module alongside secondary metabolite biosynthesis genes, and other defense responses. Co-expression and gene regulatory networks independently predicted several D clade ethylene response factors to be early regulators of the defense transcriptional module, as well as other transcription factors both known and novel in pathogen defense, including several JA-associated genes. In contrast, the strongly virulent isolate elicited a much weaker response, and a separate transcriptional module bereft of hormone signaling.ConclusionsOur findings have predicted major defense regulators and several targets for downstream functional analyses. Combined with our improved gene functional annotation, they suggest that defense is achieved through induction of Alternaria-specific immune pathways, and susceptibility is mediated by modulating hormone responses. The implication of multiple specific clade D ethylene response factors and upregulation of JA-associated genes suggests that host defense in this pathosystem involves ethylene response factors to modulate jasmonic acid signaling.

Antibacterial and antibiofilm properties of cinnamon essential oil on Pseudomonas tolaasii and application of potato starch/CEO active pads in preservation of mushroom (Agaricus bisporus)

Pseudomonas tolaasii (P. tolaasii) is considered to be the main pathogen of brown spot disease in mushrooms. Cinnamon essential oil (CEO), a natural bacteriostatic agent, has potential application prospects in the field of food preservation. The purpose of this study was to explore the antibacterial mechanism of CEO on P. tolaasii, and to prepare an active pad to study the preservation effect of mushroom, so as to provide a basis for the development of natural antibacterial agents. P. tolaasii was isolated from browning mushroom and identified by 16S rRNA gene sequencing. The antibacterial and antibiofilm activities of cinnamon essential oil (CEO) on P. tolaasii and its application in mushrooms were investigated. The results indicated that CEO had a significant inhibitory effect on P. tolaasii. After the CEO treatment, the surface of P. tolaasii cells was damaged, and significant leakage of entocyte was observed. In addition, CEO could effectively induce oxidative stress and lipid peroxidation in P. tolaasii cells, and cause irreversible damage to cell membranes, leading to energy and respiratory metabolism disorders. The activity of ROS defense enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were significantly inhibited (P < 0.05), aggravating the oxidative damage to P. tolaasii. The crystal violet staining assay and scanning electron microscopy revealed that CEO treatment not only inhibited biofilm formation, but also effectively eradicated mature biofilm. The application effects displayed that the active pads packing containing CEO had excellent antibacterial effect and maintain the quality of mushroom (Agaricus bisporus). The results of this study will provide references for further research and application of natural food antibacterial agents.

Действие органоминерального комплекса «Белый Жемчуг» на качество и выход товарных плодов яблони после длительного хранения

Biological fertilizers improve fruit quality; for instance, organomineral, or activated mixes are known to increase consumer qualities of apples. This article describes the effect of a new activated complex (non-root treatment) on the yield, chemical composition, and physiological disorders, e.g., scald and brown spot, of commercial apples after long-term storage. The research featured the natural activated complex of the Beliy Zhemchug (White Pearl) brand. The apple trees were divided into two groups: 1 – untreated control trees; 2 – trees that underwent a non-root treatment with the activated complex (1% solution of its Antifreeze variant + 1% solution of the Drip Ca + Mg variant). The trees belonged to the Sinap Orlovskiy cultivar, which is highly susceptible to brown spot and scald, both in the orchard and during long-term storage. The apples were studied for chemical composition and consumer qualities after long-term storage in a CV114-S refrigerator at +2°C and a relative humidity of 85–90%. The non-root treatment of apple trees with the Beliy Zhemchug activated complex had no significant effect on the content of soluble solids, total sugars, titrated acids, and ascorbic acid in ripe apples. Weather conditions affected the chemical composition 30 days before harvesting. The non-root treatment increased the yield of commercial apples by 1.62 times after long-time storage. The scald, brown spot, and moniliosis damage decreased by 1.9, 2.6, and 1.9 times, respectively. The activated fertilizers of the Beliy Zhemchug brand proved highly efficient as additional techniques in common apple cultivation and could be recommended to improve the quality and yield of commercial fruits after long-term storage.

Progressive alterations in mineral contents in citrus genotypes toward Alternaria citri causing brown spot of citrus.

Brown spot of citrus caused by Alternaria citri is one of the emerging threats to the successful production of citrus crops. The present study, conducted with a substantial sample size of 50 leaf samples for statistical reliability, aimed to determine the change in mineral content in citrus leaves after brown spot disease attack. Leaf samples from a diverse range of susceptible citrus varieties (Valentia late, Washington navel, and Kinnow) and resistant varieties (Citron, Eruka lemon, and Mayer lemon) were analyzed. Significant variations (p ≤ 0.05) in mineral contents were observed across reaction groups (inoculated and un-inoculated), types (resistant and susceptible), and varieties of citrus in response to infection of Alternaria citri. The analysis of variance showed significant changes in mineral levels of citrus leaves, including nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), sodium (Na), iron (Fe), and copper (Cu). The results indicate that the concentration of N and P differed by 6.63% and 1.44%, respectively, in resistant plants, while susceptible plants showed a difference of 6.07% and 1.19%. Moreover, resistant plants showed a higher concentrations of K, Ca, Mg, Zn, Na, Fe, and Cu at 8.40, 2.1, 1.83, 2.21, 1.58, 2.89, and 0.36 ppm respectively, compared to susceptible plants which showed concentrations of 5.99, 1.93, 1.47, 1.09, 1.24, 1.81, and 0.31 ppm respectively. Amounts of mineral contents were reduced in both resistant as well as susceptible plants of citrus after inoculation. Amount of N (8.56), P (1.87) % while K (10.74), Ca (2.71), Mg (2.62), Zn (2.20), Na (2.08), Fe (3.57) and Cu (0.20) ppm were recorded in un-inoculated group of citrus plants that reduced to 3.15 and 0.76% and 3.66, 1.40, 0.63,0.42, 0.74, 1.13 and 0.13 ppm in inoculated group respectively. It was accomplished that susceptible varieties contained lower ionic contents than resistant varieties. The higher concentrations of ionic contents in resistant citrus varieties build up the biochemical and physiological processes of the citrus plant, which help to restrict spread of pathogens. Further research could explore the interplay between mineral nutrition and disease resistance in citrus, potentially leading to the development of new disease-resistant varieties.

Phenylalanine Butyramide: A Butyrate Derivative as a Novel Inhibitor of Tyrosinase.

Metabolites resulting from the bacterial fermentation of dietary fibers, such as short-chain fatty acids, especially butyrate, play important roles in maintaining gut health and regulating various biological effects in the skin. However, butyrate is underutilized due to its unpleasant odor. To circumvent this organoleptic unfavorable property, phenylalanine butyramide (PBA), a butyrate precursor, has been synthesized and is currently available on the market. We evaluated the inhibition of mushroom tyrosinase by butyrate and PBA through in vitro assays, finding IC50 values of 34.7 mM and 120.3 mM, respectively. Docking calculations using a homology model of human tyrosinase identified a putative binding mode of PBA into the catalytic site. The anti-aging and anti-spot efficacy of topical PBA was evaluated in a randomized, double-blind, parallel-arm, placebo-controlled clinical trial involving 43 women affected by photo-damage. The results of this study showed that PBA significantly improved skin conditions compared to the placebo and was well tolerated. Specifically, PBA demonstrated strong skin depigmenting activity on both UV and brown spots (UV: -12.7% and -9.9%, Bs: -20.8% and -17.7% after 15 and 30 days, respectively, p < 0.001). Moreover, PBA brightened and lightened the skin (ITA°: +12% and 13% after 15 and 30 days, respectively, p < 0.001). Finally, PBA significantly improved skin elasticity (Ua/Uf: +12.4% and +32.3% after 15 and 30 days, respectively, p < 0.001) and firmness (Uf: -3.2% and -14.9% after 15 and 30 days, respectively, p < 0.01).

Aplikasi Ekstrak Mimosa invisa L. dan Pupuk NPK Dosis Rendah dalam Pengendalian Hama dan Patogen Padi Gogo

This study aimed to determine the effect of Mimosa invisa L. weed extract application and a low dose of NPK fertilizer on the intensity of pest attack and pathogen infection on upland rice. This research has been done using Split Plot Design with 3 replications. The main plot was the dose of NPK fertilizer (50% and 100% NPK). The subplot was the concentration of weed extract (0, 1.25, 1.67, 2.5, and 5% concentrations). The variables observed include the intensity of grasshopper and case worm attack, Cercospora sp. that causes brown spots, and Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight. The results showed that the application of weed extract under a low dose (50% NPK) has not been able to suppress the intensity of grasshopper and case worm attacks, and the intensity of the pathogen causes brown spots and bacterial leaf blight. However, 2,5% and 5% concentrations of weed extract can potentially suppress pathogen infection that causes brown spots on upland rice. Keywords: grasshopper, brown spots, Mimosa invisa L.weed extract, case worm, bacterial leaf blight, NPK fertilizer

First Report of Leaf Brown Spot Caused by Diaporthe phoenicicola on Pachira glabra in China.

Pachira glabra is an increasingly important ornamental landscape tree in southern China. In August 2022, brown spots were observed on P. glabra leaves in Xiangtan City, Hunan Province, China (27.932°N, 113.020°E), affecting up to 40% of the 792 trees surveyed. On each diseased tree, nearly 30% leaves had symptoms, with an average severity of 21.2 ± 5.8% (n=100). The disease initially started as small yellow lesions along leaf margins, which later progressed to pale brown to brown with dark brown borders, eventually coalescing into large necrotic areas. Thirty symptomatic leaf samples (2 × 2 mm) were surfaced-sterilized in 75% ethanol for 10 s, 2% NaOCl for 30 s, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and incubated at 25°C for 5 to 7 days in dark. Eight morphologically similar isolates were obtained from diseased leaf samples through single-spore isolation. On PDA, colonies initially appeared white, turning gray, while the reverse developed a pale yellowish hue. Aerial mycelia were white, cottony, and developed visible black pycnidia with oil droplets at maturity. The α-conidia were unicellular, hyaline, aseptate, oval or fusiform, usually with 1 or 2 guttule(s) and rounded at each end. These conidia were 5.3-8.6 × 1.7-2.5 μm (avg. 6.7 × 2.2 μm, n = 100) and present more frequently than β-conidia.The β-conidia were unicellular, hyaline, aseptate, filiform, straight or hamate, eguttulate, 14.6-23.3 × 0.4-1.3 μm (avg. 18.4 × 0.9 μm, n = 30). Morphologically, the fungi were identified as Diaporthe sp. (Udayanga et al. 2014). For molecular identification, the internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), calmodulin (CAL), tubulin 2 (TUB2), and histone H3 (HIS3) sequences of all isolates were amplified from genomic DNA, using primers ITS4/ITS5 (White et al. 1990), TEF-2/728F and CALD-38F/CALD-752R (Carbone and Kohn 1999), Bt2a/Bt2b and H3-1a/H3-1b (Glass and Donaldson 1995; Crous et al. 2004), respectively. The GenBank accession numbers for a representative isolate gpg2023-1 were OR533573 (ITS), OR570887 (EF1-α), OR570888 (TUB2), OR570890 (CAL), and OR570889 (HIS3). BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences were 99%, 99%, 99%, 99%, and 98% identity, respectively, with those of Diaporthe phoenicicola (GenBank: KC343032.1, KC343758.1, KC344000.1, KC343516.1, and KC343274.1). To confirm the pathogen's identity, phylogenetic analysis using MEGA7.0 based on Maximum Likelihood was constructed. Isolate gpg2023-1 clustered with D. phoenicicola. Based on morphological and molecular data, the fungus was identified as D. phoenicicola. Next, pathogenicity tests were performed three times on one-year-old potted P. glabra plants. For each isolate, twelve healthy leaves on each of three plants were either wounded by a sterile needle or left unwounded, and then sprayed with a conidial suspension (1×106 conidia/ml) for each isolate. Control plants received with sterile water only. Plants were kept in a greenhouse at 25°C, 80% relative humidity, with a 12-h photoperiod. All wounded, inoculated leaves developed brown spot symptoms similar to those observed in the field with six days, while unwounded leaves and control plants remained symptom-free. The fungus was reisolated from all diseased leaves, fulfilling Koch's postulates and proving D. phoenicicola as the causative agent of this brown spot disease on P. glabra. While D. pachirae has been reported to cause leaf spot on P. glabra in Brazil (Milagres et al. 2018), this study marks the first report of D. phoenicicola causing leaf brown spot on P. glabra in China. This finding can help develop control strategies for this disease.

First Report of Leaf Rot Caused by Apiospora paraphaeosperma on Lily in Wuhan, China.

Lily (Lilium spp.) is a valuable ornamental bulb flower plant in Liliaceae, and its bulbs have high edible and medicinal value. Compared with bulb propagation of other lilies, seed propagation and short growth period are the most significant characteristics of Lilium×formolongi. In 2023, leaf rot disease (LRD) was observed on approximately 70% of the Lilium×formolongi seedlings sown in an experimental greenhouse in Wuhan, Hubei province, China. Irregular brown water-soaked spots were discovered in the early stages of infected seedlings. Then, spots spread throughout the leaves and caused the leaves to brown, soften, and wilted. A pathogen associated with symptoms was isolated by incubating sterilized leaves on potato dextrose agar plates at 25 ℃ for 2-3 days. Then, a pure single colony was isolated through a single hyphal tip isolation method. The fungal colony was white with abundant aerial mycelium and produced a yellow pigment diffusible into the agar. Microscopically, isolated mycelia were reticulate and pale yellow, while conidia were dark brown, smooth, and spherical, 7.31 to 6.98 × 4.03 to 3.87μm (average 5.44×5.41μm; n=30); oval in lateral view, and had a light stripe in the middle. To identify the species of the fungus at the molecular level, ITS and EF-1α genes were amplified and sequenced using primers ITS1/ITS4 (M Gardes et al. 1993) and 758F/986R (Carbone and Kohn 1999). The BLAST results in GenBank showed that the ITS(OR523578) and EF-1α(PP066842) sequences of LRD shared 99.82% and 99.24% identity with the distinct Apiospora paraphaeosperma strains (GenBank accession MT040110, ON806628.1, respectively). Combined with the morphology of the colony and conidium, the fungus was identified as Ap. paraphaeosperma. In the pathogenicity test, six healthy leaves were inoculated with mycelium disc and then kept in an incubator (22 ℃, 90% humidity, 16h light /8h darkness). The inoculated leaves showed necrosis and wilt symptoms similar to those observed in the greenhouse, while the control leaves were asymptomatic. A re-isolation, morphology identification and DNA sequencing of the fungus confirmed its infection with Ap. paraphaeosperma in Lilium spp. At present, rot caused by Ap. paraphaeosperma has only been reported in Thailand and South Korea, both of which are found on bamboo stems (Hyde et al. 2016; Sun Lul Kwon et al. 2022). As far as we know, this is the first report of leaf rot of lily caused by Ap. paraphaeosperma in China. This report can help identify this disease and further develop effective control measures.

Bisifusarium lunatum causing cladode soft rot in pear cactus (Nopalea cochenillifera L. Salm-Dyck) in Brazil.

Cactus pear var. miúda (Nopalea cochenillifera L. Salm-Dyck) is an important crop for the Northeast region of Brazil, composing one of the main sources of animal feed. By April 2021, cladode rot caused death of several cactus pear plants in a production area located in Itaporanga, Paraíba state, Brazil (7°21'55.35" S and 38°11'38.68" W). The infected cladodes showed brown circular necrotic spots, and soft rot with perforations that extended throughout the cladode, followed by tipping over and death of the infected plants. The incidence of the disease ranged from 10 to 30% of the plants. Bisifusarium strains were isolated and cultured on potato dextrose agar (PDA) and syntetic-nutrient-poor-agar (SNA). The colonies showed purple color on PDA. On SNA, macroconidia (n = 100) were abundant, hyaline, slightly falcate, three-septate, measuring 11.0-23.1 x 2.3-4.1 μm. Microconidia (n = 100) were oval, generally aseptate, measuring 4.1-8.7 x 2.3-3.0 μm. Conidiogenic cells formed into short monophialides. Chlamydospores were not observed. According to these morphological features, the pathogen was initially identified as Bisifusarium lunatum (Gryzenhoutm et al. 2017). For further confirmation of the identification, the partial sequences of translation elongation factor 1-alpha (TEF1-α) and the second largest subunit of RNA polymerase II (RPB2) genes were sequenced for a representative isolate (CMA 34: GenBank accession no: TEF1-α: OR536502; and RPB2: OR553509) and compared to other Bisifusarium species from GenBank database. Subsequently, it was subjected to a phylogenetic analysis of maximum likelihood including previously published sequences. According to BLAST searches, the TEF1-α and RPB2 sequences were 99% (637/640 nt) and 100% (312/312 nt) similar to B. lunatum (COUFAL0213: TEF1-α (MK640219), and RPB2 (MK301291)), respectively. The isolate was also clustered in a clade containing the ex-type of B. lunatum with 100% support (SH-aLRT and UFboot), being confidently assigned to this species. The pathogenicity test was performed after Medeiros et al. (2015), by using healthy two months old cactus pear seedlings (n = 10) cultivated in a greenhouse. Sterile toothpicks were distributed over colonies of the representative isolate grown on PDA at 25 ± 2 °C for seven days. Seedling cladodes were stuck with the toothpicks, moistened with sterile water and covered with transparent plastic bags for 24h, thus simulating a humid chamber. Following three months, all control plants (stuck with sterile toothpicks) remained healthy, while those inoculated with the representative isolate exhibited rot symptoms. This test was performed twice. B. lunatum was reisolated from symptomatic cladodes and identified as previously described, thus fulfilling the Koch's postulates. To our best knowledge, this is the first report of B. lunatum causing soft rot on N. cochenillifera in Brazil. Besides N. cochenillifera, this species was also reported on Opuntia ficus-indica in India (Gryzenhoutm et al., 2017), which raises concern regarding its ability to infect other forage sources for cattle feed in Brazilian semiarid regions. The present study highlights that the precise identification of B. lunatum is a key factor to adjust control strategies and management of the disease to prevent the spread of this disease to prevent its spread to other crops.

First report of leaf blight caused by Nigrospora hainanensis on Euonymus japonicus in China.

Euonymus japonicus Thunb., belonging to the family Celastreace and native to East Asia, is a widely cultivated evergreen ornamental woody plant with important ecological and economic values. In May 2023, serious leaf blight of E. japonicus occurred in the campus green space at Guiyang University, Guizhou Province, China (26°55'85"N, 106°78'04"E). Early symptom appeared as small, circular light brown spots on the edges or tips of the leaves. Then, the spot developed visible necrosis, initially light brown to dark brown halos with clear margins. Subsequently, severely infected leaves appear totally wilt, and significantly decrease their ornamental values. In a 0.07-ha field, the disease incidence reached to 40-55%. To identify the pathogen, ten typical symptomatic E. japonicus leaves were collected. They were initially immersed in 75% ethanol for 3 min, and by sodium hypochlorite (4% NaClO) solution for 45 s, and ultimately rinsed with sterile distilled water (dH2O) five times for not less than 1 min each time, then, placed the leaves on potato dextrose agar (PDA) medium and cultured for 5 days at 25°C in constant temperature incubator. Cultures were purified to yield eight isolates. Early colonies are white and regularly rounded, gradually turning dark brown to black with fluffy mycelium. Conidia were single celled, smooth, black, spherical or ellipsoidal. The conidia size of the representative strain, GY-2 and GY-3, was averagely 12.3-17.3 µm × 10.8-17 µm (n = 50). The conidiogenous cells were monoblastic, hyaline, globose or ampulliform. Morphology-based identification revealed the strain as Nigrospora spp. (Wang et al., 2017). For further confirmation, PCR of GY-2 and GY-3 DNA was performed with the primers ITS1/ITS4 (White et al., 1990), Bt2a-F/Bt2b-R (Glass and Don-aldson 1995), and TEF1-728F/TEF1-986R (Carbone and Kohn 1999). Sequences of the ITS region, TUB and TEF1 genes from the strain GY-2 and GY-3 were deposited in GenBank. (GY-2: OR999377, PP112221 and PP150467; GY-3: PP406871, PP421045 and PP421046, respectively). BLAST analysis showed GY-2 100%, 100%, and 98.36%; GY-3 99.43%, 98.21% and 100% (ITS region, TEF1, and TUB) identity to N. hainanensis sequences (accession numbers. NR_153480.1, KY019415.1, and KY019464.1; KX986094.1, OP611475.1, and KY019597.1). Additionally, tandem sequences of ITS, TUB and TEF1 constructed by MEGA 7.0 confimed the homology through the phylogenetic tree. Pathogenicity tests were conducted on healthy plants grown, each 5 mm diameter of active growing mycelium plug of isolate GY-2 was attached to 15 leaves from five healthy 2-year-old E. japonicu plants. The same number of leaves in the control group were treated with non-inoculated plugs only. All the plants were incubated at 25°C and 75% relative humidity with a 16-h/8-h photoperiod. After 10 days, no symptoms appeared on the leaves of the control group. In contrast, symptomatic blight appeared on all leaves inoculated with GY-2. Pathogenicity tests were performed five times. Pure strains were re-isolated from diseased leaves and, confirmed to be N. hainanensis based on the above methods. Recently, Nigrospora oryzae was reported as causal agent of leaf spots on Euonymus japonicus in China (Xu et al., 2023). To our knowledge, this study is the first report of N. hainanensis causing leaf blight on E. japonicu. Identification of the etiological agent may provide assistance for sustainable management in the future.

First record of Canthesancus gulo Stal, 1863 (Reduviidae, Stenopodainae) from Maharashtra, India

The brownish castaneous spot assassin bug Canthesancus gulo Stal, 1863 is reported from Maharashtra for the first time, as the first species of this genus in the Maharashtra state. The diagnostic characters, colour images, and current geographical distribution of species are given.

‘Sultanina’ leaves increase their trehalose content in response to grapevine brown leaf spot infection by regulating the pentose and glucuronate interchange pathway

Since the first discovery of grapevine brown leaf spot disease in Turpan, Xinjiang, China, in 2007, it has been a common occurrence in grapevine-growing regions. Grapevine brown leaf spot seriously decreases fruit yield and has become one of the most important leaf diseases in the Turpan region. However, thus far, there have been no reports on the evaluation of grapevine germplasm resources for resistance to brown leaf spot disease. In addition, the response mechanism of grapevine leaves to brown leaf spot infection has not been revealed. To better understand the resistance of grapevine germplasm resources to brown leaf spot disease, we first evaluated resistance in grapevine varieties. On this basis, the most susceptible variety ‘Sultanina’ was selected as the experimental material for this study. Transcriptome analysis and carbohydrate content analysis were performed on ‘Sultanina’ leaves with different levels of disease susceptibility. As the severity of the disease increased, the content of fructose gradually decreased, while the content of trehalose gradually increased. Transcriptome data revealed that differentially expressed genes were enriched in the pentose and glucuronate interchange pathway. These results suggest that the sugar trehalose may play an important role in the response of ‘Sultanina’ leaves to brown leaf spot infection. In addition, the pentose and glucuronate interchange pathway may be involved in the response mechanism of brown leaf spot disease. Our work provides new insights into the mechanism of the ‘Sultanina’ leaf response to brown leaf spot infection.

First report of Lasiodipilodia theobromae Causing leaf spot of Agave sisalana in GuangXi, China.

Agave sisalana, as an excellent fiber producing plant, is mainly planted in Guangxi Province, China. In November 2023, a foliar disease occured on A. sisalana at Liangjiang Town (108.3593 W, 23.4723 N), Wuming District, Nanning in GuangXi, China. Approximately 50 to 60% of the plants (n=200) had obvious leaf spots on more than 70% of the leaves. On the leaves of sisal, circular or irregularly shaped yellow brown spots can be seen, sunken, with no halo on the edges. As time goes on, the lesion gradually expands to the entire blade of the sword (Figure 1A, 1B). To identify the disease etiology, ten agave leaves were collected from GuangXi. Symptomatic midribs were cut into 3×3 mm pieces, surface sterilized with 75 % ethanol for 20 s, rinsed with sterilized distilled water three times, air dried on sterile filter paper, plated on photo dextrose agar (PDA) medium, and incubated at 28 ℃ in the dark. Five isolates (JM01, JM02, JM03, JM05, JM06) with similar morphology were obtained. Colonies on PDA medium were white to grayish-white with atrial mycelia growing initially upward and then forming clusters (Figure 1E). After five days, mycelia turned grayish black. Immature conidia were initially hyaline, aseptate, and ellipsoid. Mature conidia were dark brown, one septate, longitudinal striate, and 22.1 to 26.3×10.2 to 14.9 μm (Figure 1F). Morphologically , the isolates were identified as Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genome DNA of five representative isolate was extracted using the Fungi Genomic DNA Purification kit. The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF-1α) and β-tublin (TUB) gene were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively, and sequenced. The ITS (PP209594), TEF-1α (PP234629), and TUB (PP234628) sequences of representative isolate JM01 were deposited in GeneBank. BLAST searches showed >99% nucleotide identity to sequences of L. theobromae (ITS, 99.26% to NR111174; TEF-1α, 99.69% to MM840490; TUB, 98.92% to MN172230). Phylogenetic analysis using maximum likelihood based on the combined ITS, TEF-1α, and TUB sequences of the isolates and reference sequences of Lasiodiplodias spp. from GenBank indicated the isolates obtained in this study formed a clade strongly supported based on bootstrap values to the ex-type isolate CBS164.96 sequences of L.theobromae (Figure 2). To test pathogenicity, JM01 was tested by inoculation leaves of one year old agave plants, the epidermis at the inoculation site, 10, 15 and 20 cm below to the crown, was wiped with a 75% alcohol cotton ball, washed three times with sterile water, and punctured (5 mm diameter) with a sterile inoculation needle. A 5 mm block of each isolate cultured on PDA for 3 days was attached to the inoculation site. Controls were inoculated with sterile PDA. The inoculation area was covered with plastic wrap. All plants were kept in a controlled greenhouse at 27℃, 80% relative humidity, and natural daylight, and watered weekly. Each treatment was repeated three times. Remove the block one day later. Three days after inoculation, all inoculated had typical symptoms,but control were healthy (Figure 1C, 1D). Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. L. Theobromae has been reported as the cause of leaf rot on A. angustifolia in Mexico (Reyes-García et al. 2023). To our knowledge, this is the first report of L. theobromae causing leaf spot on A. sisalana in GuangXi, China. L. theobromae is primarily a plant pathogen that causes rotting and dieback in fruits and plants in tropical and subtropical regions (Puttanna 1967). This study is useful to focus on management strategies for leaf rot disease by L. theobromae of A. sisalana.

Highly Accurate and Lightweight Detection Model of Apple Leaf Diseases Based on YOLO

To mitigate problems concerning small-sized spots on apple leaves and the difficulties associated with the accurate detection of spot targets exacerbated by the complex backgrounds of orchards, this research used alternaria leaf spots, rust, brown spots, gray spots, and frog eye leaf spots on apple leaves as the research object and proposed the use of a high-accuracy detection model YOLOv5-Res (YOLOv5-Resblock) and lightweight detection model YOLOv5-Res4 (YOLOv5-Resblock-C4). Firstly, a multiscale feature extraction module, ResBlock (residual block), was designed by combining the Inception multi-branch structure and ResNet residual idea. Secondly, a lightweight feature fusion module C4 (CSP Bottleneck with four convolutions) was designed to reduce the number of model parameters while improving the detection ability of small targets. Finally, a parameter-streamlining strategy based on an optimized model architecture was proposed. The experimental results show that the performance of the YOLOv5-Res model and YOLOv5-Res4 model is significantly improved, with the mAP0.5 values increasing by 2.8% and 2.2% compared to the YOLOv5s model and YOLOv5n model, respectively. The sizes of the YOLOv5-Res model and YOLOv5-Res4 model are only 10.8 MB and 2.4 MB, and the model parameter counts are reduced by 22% and 38.3% compared to the YOLOv5s model and YOLOv5n model.

First Report of Exserohilum rostratum Causing Brown Spot on Spartina alterniflora in China.

Spartina alterniflora Loisel, a perennial grass, has become an invasive species in China's coastal wetlands (Zhang et al. 2018). In July 2021, brown spot symptoms were observed on S. alterniflora in a coastal wetland (21°45'48″N, 108°44'00″E) in Beihai City, Guangxi Province, China. The disease affected approximately 50% of the plants in the surveyed area (0.2 ha) and was also observed in other regions of Beihai. It caused brown lesions with a gray or whitish center on the leaves and stems of S. alterniflora. As the disease developed, it ultimately led to leaf shedding and plant death. To isolate the causal agent, 18 fragments (~ 5 mm) from six symptomatic plants (3 leaf pieces per plant) were surface sterilized with 1% NaOCl solution for 2 min and rinsed three times with sterilized water. Subsequently, the tissues were placed on potato dextrose agar (PDA) medium supplemented with chloramphenicol (0.1 g/liter) and incubated at 28°C for three days. The hyphal tips were transferred onto fresh PDA to obtain pure cultures. A total of 25 isolates were obtained, 20 of which shared similar morphologies, while the remaining five exhibited distinct morphological characteristics and were non-pathogenic to S. alterniflora. Three isolates (MC16.1.3, MC16.6.2, and MC16.8.3) were randomly selected from the 20 for further investigation. The colonies on PDA were flat with dense aerial mycelia. The colony margins were entire, light brown in the centre, white to grey at the margin; reverse dark brown in the centre, gray at the margin. Conidia were straight to slightly curved, light olive-brown to dark olive-brown, septate, measured 33.5 to 79.1 μm × 10.4 to 18.7 μm (average 52.9 × 14.4 μm, n = 100), with a distinctly protruding hilum swelled from the basal cell. For molecular identification, the genomic DNA was extracted from mycelium on PDA using the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1 alpha (TEF1-α) genes were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1999), and EF1-983/EF1-2218 (Rehner et al. 2005), respectively. A BLAST analysis revealed that the ITS (OR516787-9), GAPDH (OR523686-8), and TEF-α (OR523683-5) had 99.1 to 99.7% identity with those of E. rostratum strains BRIP 11417 (LT837836, LT882553, and LT896656) and CBS 128061 (KT265240, LT715900, and LT896658) (Hernández-Restrepo et al. 2018). Based on the concatenated sequences, a phylogenetic tree generated by PhyloSuite software (Zhang et al., 2020) through Bayesian inference (BI) and Maximum Likelihood (ML) methods placed the isolates within E. rostratum. These morphological characteristics and molecular analyses confirmed the pathogen as E. rostratum (Hernández-Restrepo et al. 2018; Kaboré et al. 2022). To confirm pathogenicity, a conidial water suspension (~ 1 × 106 conidia/ml) of each of the three strains was inoculated on nine healthy S. alterniflora plants that had been grown for six months. Control plants were treated with sterile water. All plants were then enclosed in plastic bags and incubated in a greenhouse at 28°C. Six days after inoculation, the plants exhibited symptoms similar to those observed in nature. The control plants developed no symptoms. These experiments were replicated three times with similar results. To fulfill Koch's postulates, E. rostratum was consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing, whereas no fungus was isolated from the control plants. In recent years, S. alterniflora has posed a serious threat to the indigenous biodiversity of wetland ecosystems (Zhang et al. 2018). To our knowledge, this is the first report of E. rostratum causing brown spot on S. alterniflora worldwide.

First report of Fusarium commune Causing Bulb Rot on Lily (Lilium brownii var. Viridulum Baker) in Hunan Province of China.

Lily (Lilium brownii var. Viridulum Baker) is a well-known edible plant with large, white and sweet bulb scales that has important medicinal value (Zhou et al. 2021) and is grown mainly in the Hebei, Shanxi and Henan provinces of China. In May 2021, a case of bulb rot was discovered in a 3.33 hm2 plantation in Huaihua, Hunan Province, affecting 20% of the area (27°59'30″N, 110°32'20″E). The disease is most severe during the rainy season in May and June. In the early stage, irregular brown spots appeared on the lily scales, the necrosis was depressed and gradually enlarges, and in the later stage, the scales were scattered from the base of the disc and slough off. Ten samples were taken randomly from different plants in the plantation area to isolate the pathogens. After washing with sterile water, they were cut into small pieces and sterilised with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s, rinsed three times with sterile water and dried on sterile filter paper, then placed on a water agar plate and incubated in the dark in a constant temperature incubator at 28℃ for 3 to 5 days. After 2 days, the mycelium at the edge of the colony was transferred to a PDA plate and incubated for 3-5 days at 28°C in the dark to obtain pure fungal isolates. Eighteen purified fungal isolates were obtained, of which sixteen looked like Fusarium (88.9% isolation rate) and three representative isolates (BHBR2, BHBR3 and BHBR5) were selected for further study. The surface of this fungus was white with dense aerial mycelium. Some had an orange centre in the medium. Microconidia were oval in shape and appeared either straight or slightly curved. These microconidia were colourless, had 0-1 septa and measured 3.334 to 14.724 × 2.216 to 5.385 μm (n=100). Macroconidia were predominantly three-septate, crescent-shaped structures that were thin-walled and slightly curved. Cells at the apex and base were similarly curved. Macroconidia measured 17.956 to 32.150 × 2.788 to 4.492 μm (n=100). The mitochondrial small subunit (mtSSU) and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the NMS1/NMS2 and TEF-R/TEF-F primers to verify the identity of the pathogens (Stewart et al. 2006). The sequences were submitted to GenBank (BHBR2: mtSSU, PP273435; TEF, OR900976; BHBR3: mtSSU, PP277729; TEF, OR900977; BHBR5: mtSSU, PP277728; TEF, OR900978). A concatenated phylogenetic tree of the two genes was constructed and analysis showed that BHBR2, BHBR3 and BHBR5 were significantly clustered with Fusarium commune. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as Fusarium commune. We carried out pathogenicity tests using two methods, one in which 6 × 6 mm fungal blocks were inoculated on lily (L. brownie var. viridulum Baker) scales and controls inoculated with sterile blocks, and the other in which strain BHBR2 was selected to carry out pathogenicity tests on bulbs of live plants soaked with 50 ml of a 1 × 106 conidial suspension and bulbs of control plants soaked with sterile water, all in three replicates. They were placed in a growth chamber at 28°C and 80% relative humidity, and the scales were moistened with moistened sterile filter paper. After 3 days of rearing treated scales, lesions appeared on lily scales inoculated with mycelial blocks and expanded with time, whereas no lesions appeared on lily scales inoculated with sterile blocks. One month later, whole plants soaked in the spore suspension wilted, while the control plants grew well. The pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. This confirms Koch's hypothesis. Fusarium commune has been shown to be the most important pathogenic fungus causing root rot in Alfalfa (Medicago sativa) (Yang et al. 2022) and blueberry (Vaccinium uliginosum L.) (Li et al. 2023) in China. To our knowledge, this is the first report of Fusarium commune causing lily bulb rot in the world, which will lay the foundation for future control of lily bulb rot.

The interaction of fungicides and nitrogen dose under two crop establishment methods influences the severity of brown spot disease in rice

Brown spot (BS) disease poses a significant threat to rice cultivation globally, impacting millions of hectares annually. Research indicates that the severity of BS is closely linked to abiotic stresses and crop nutrition. A field experiment was conducted using transplanted (TP) and direct-seeded-rice (DSR) crop establishment methods to optimize the effective fungicide and nitrogen dose. The split-plot design was applied to study the effect of two factors (N-dose and fungicides) varying at different levels under two different crop establishment methods (TP and DSR) for two consecutive Kharif seasons (2021 and 2022). The BS severity was measured as the percent disease index (PDI), and the results of two seasons were pooled. The results indicated that all three factors significantly (p = 0.05) influenced the PDI and grain yield. The PDI under different fungicides was higher in DSR (30.20–88.90) than in TP rice (21.64–70.00). Similarly, increased N-dose altered the PDI negatively (p = 0.05) in both TP (32.16–46.12) and DSR (44.35–59.09) methods. The results indicated Tebuconazole 50% + Trifloxystrobin 25% WG (T7) at 125% recommended dose of nitrogen as the best combination, as it recorded the lowest PDI of 21.6 and 30.2 in the TP and DSR, respectively. Similarly, all three factors interacted significantly (p = 0.05) and affected the final grain yield. Overall, the BS is more severe in DSR, and a higher dose of N reduces the BS severity in both TP and DSR. Therefore, N fertilizer management, especially in the DSR, is essential, along with a fungicide, for effective management of the brown spot disease.