Brown Leaf Research Articles

Transcriptome profiling uncovers the involvement of CmXyn1, a glycosyl hydrolase 11, in Cochliobolus miyabeanus pathogenicity

Necrotrophic pathogen Cochliobolus miyabeanus (C. miyabeanus) causes rice brown leaf spot disease and drastically affects the yield and quality of rice grains. However, the molecular mechanism of rice-C. miyabeanus remains poorly understood due to the limited research conducted on this pathosystem. To elucidate the molecular mechanism of rice-C. miyabeanus, a transcriptome analysis was conducted from in vitro and in planta grown C. miyabeanus. This analysis led to the identification of a total of 24,060 genes of which 426 in vitro and 57 in planta expressed genes were predicted to encode for secretory proteins. As these 57 genes were specifically expressed in planta and were predicted to be secretory in nature, these were consider as putative effectors, highlighting their possible roles in the fungal pathogenicity. Notably, among these putative effectors, CmXyn1 which encodes a glycosyl hydrolase 11 displayed the highest expression level under in planta conditions and was thus selected for further functional characterization. Interestingly, the extracellular expression of CmXyn1 transiently induced cell death in Nicotiana benthamiana leaves, while intracellular expression was comparatively lesser effective. In addition, transcriptome analysis on rice leaves during C. miyabeanus infection and comparing it to the rice leaf transcriptome data obtained during hemibiotrophic pathogen Magnaporthe oryzae infection led to the discovery of 18 receptors/receptor-like kinases that were commonly expressed in response to both pathogens, indicating their key roles in rice defense response. Taken together, our findings provide new insights into rice-C. miyabeanus interaction as well as the unique and common defense responses of rice against hemibiotroph and necrotroph model systems.

Leaf spot of Alnus cremastogyne Caused by Colletotrichum gloeosporioides in Sichuan, China.

Alnus cremastogyne Burk, a broad-leaved tree endemic to south-western China, has both ecological and economic value. The tree is widely used in furniture, timber, windbreaks and sand fixation, and soil and water conservation (Tariq et al. 2018). In December 2020, a new leaf spot disease was discovered on A. cremastogyne in two plant nurseries in Bazhong City (31°15' to 32°45N, 106°21' to 107°45'E), with 77.53% disease incidence. Among the infected trees, 69.54% of the leaves were covered with symptoms of the disease. The typical symptoms initially appeared as irregular brown necrotic lesions, while some lesions were surrounded by a light yellow halo. As the disease progressed, the number of necrotic lesions increased, and lesions gradually expanded and coalesced (Fig. 1). Finally, the disease caused the leaves of A. cremastogyne to wither, curl, die, and fall off. Ten symptomatic leaves were collected from 5 different trees in the two plant nurseries. The leaves with symptoms of leaf spot disease were collected and cut from the junction between the diseased and the healthy tissues. The infected tissues from 10 samples were cut into small 2.5 × 2.5 mm pieces. Infected tissues was sterilized in 3% NaClO solution for 60 s followed by 75% ethanol for 90 s, rinsed three times in sterile water, blot-dried with autoclaved paper towels, and then cultured on potato dextrose agar (PDA) at 25℃ for 4 to 8 days in 12 h/12 h light/dark conditions. After 8 days, the colony diameter reached 71.2 to 79.8 mm. The colonies were initially light pink, and then turned white with pale orange beneath. The conidia were single-celled, aseptate, colorless, cylindrical, straight, bluntly rounded at both ends, and measured 11.6 to 15.9 × 4.3 to 6.1 μm (n = 100). These morphological characteristics were consistent with the description of Colletotrichum gloeosporioides (Pan et al. 2021). For molecular identification, the genomic DNA of a representative isolate, QM202012, was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone & Kohn, 1999) and GDF/GDR (Templeton et al. 1992), respectively. Sequences were deposited in GenBank (ITS: OL744612, ACT: OL763390, and GAPDH: OL799166). BLAST results indicated that the ITS, ACT, and GAPDH sequences showed >99% identity with C. gloeosporioides sequences in NCBI (GenBank NR160754, MG561657, and KP145407). Identification was confirmed by Bayesian inference using Mr Bayer (Fig 2) A conidial suspension (1 × 106 conidia/ml) was used to test pathogenicity on the leaves of 4-year-old A. cremastogyne plants (10 plants). Fifteen leaves of each plant (10 pots in total) were inoculated with the spore suspension on the leaves. The same number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under 16 h/8 h photoperiod and 67 to 78% relative humidity. The symptoms observed on the inoculated plants were similar to those of the original diseased plants, with 100% of the inoculated plants being infested with brown leaf spots, but the controls remained symptom-free. C. gloeosporioides was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, showing similar results each time, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot on A. cremastogyne caused by C. gloeosporioides in China. This finding indicates that C. gloeosporioides may become a serious threat to A. cremastogyne production in Bazhong City and helps to further examine and prevent leaf spot disease in A. cremastogyne growing areas in Bazhong City.

Comparative transcriptomics provides insights into the pathogenic immune response of brown leaf spots in weeping forsythia.

Weeping forsythia is an important ornamental, ecological, and medicinal plant. Brown leaf spots reduce the large-scale production of weeping forsythia as a medicinal crop. Alternaria alternata is a pathogen causing brown leaf spots in weeping forsythia; however, its pathogenesis and the immune response mechanisms of weeping forsythia remain unclear. In this study, we identified the two mechanisms based on morphological anatomy, physiological indexes, and gene expression analyses. Our results showed that A. alternata induced leaf stomata to open, invaded the mesophyll, dissolved the cell wall, destroyed the cell membrane, and decreased the number of chloroplasts by up-regulating the expression of auxin-activated signaling pathway genes. A. alternata also down-regulated iron ion homeostasis and binding-related genes, which caused an increase in the levels of iron ions and reactive oxygen species in leaves. These processes eventually led to programmed cell death, destroying palisade and spongy tissues and causing the formation of iron rust spots. A. alternata also caused defense and hypersensitive responses in weeping forsythia through signaling pathways mediated by flg22-like and elf18-like polypeptides, ethylene, H2O2, and bacterial secretion systems. Our study provides a theoretical basis for the control of brown leaf spots in weeping forsythia.

Effects of Biochar on the Cd Uptake by Rice and the Cd Fractions in Paddy Soil: A 3-Year Field Experiment

Biochar is a potential amendment for the remediation of Cd-contaminated soils. Although the immobilization effect of biochar on soil Cd has been studied under indoor laboratory conditions, the effect of biochar on rice Cd uptake and soil Cd fractions under field conditions is still poorly understood. Here, the Cd content of the different organs of rice and the Cd fractions in soil were characterized for three years after the application of different amounts of biochar (0, 7.5, 15, 30 t ha−1, and 3 t ha−1 year−1). The Cd content of brown rice, husk, leaf, stem and sheath, and root under biochar treatment could be maximally reduced by up to 26.25%, 20.16%, 20.74%, 33.2%, and 26.89%, respectively. Biochar altered the Cd fractions in soil, including the decrease in exchangeable Cd content and the increase in Fe-Mn oxide bound Cd and organic bound Cd. The concentration factor of Cd uptake by rice was reduced by 32% under biochar application, while biochar had little influence on the transfer factor and distribution factor. The immobilization effect of biochar on soil Cd lasted for at least three years, but the trend of Cd immobilization efficiency over time for different amounts of biochar treatment was different. The Risk Assessment Code (RAC) of Cd in soil with biochar amendment could be reduced to a medium risk level from a high risk level. Redundancy analysis (RDA) revealed that changes in soil pH and Fe-Mn oxide bound Cd content caused by biochar application contributed most to the reduction in the Cd content of rice organs. These findings would enhance our understanding of the immobilization effect of biochar on Cd in paddy soil under field conditions.

First Report of Rose Apple Leaf Spot Caused by Colletotrichum siamense in Thailand.

The rose apple (Syzygium samarangense (Blume) Merr. & L.M.Perry) plant has been commonly cultivated in Thailand. In May of 2022, leaf spot disease of rose apple was discovered in Chiang Mai Province, Thailand, with approximately 30% disease incidence. The typical symptoms initially showed brown spots (0.1 to 0.5 mm in diameter) with a yellow halo surrounding. These spots then expanded with black edges and the infected leaves appear blighted and desiccated. In humid conditions, pale yellow conidiomata formed on the lesions. Small pieces (5 × 5 mm2) of the margins between lesions and the healthy tissue were surface disinfected with 1% NaClO for 1 min, 70% ethanol for 30 s, and washed three times with sterile distilled water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25 ºC for three days. Three fungal isolates (SDBR-CMU419, SDBR-CMU420, and SDBR-CMU421) were obtained that exhibited similar morphology. Fungal colonies appeared white to gray with cottony mycelia after incubation on PDA at 25 ºC for one week. All fungal isolates produced asexual morph on PDA. Setae were 5590 × 2.53.5 µm, brown with 13-septa, cylindrical base, and tip rounded. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 2050 µm long (n = 50). Conidia were one-celled, hyaline, smooth-walled, aseptate, straight, cylindrical, end round, guttulate, 1017 × 35 µm (n = 50). Appressoria were mostly formed from mycelia, oval to irregular, brown to dark brown, smooth-walled, 610 × 57 µm (n = 50). Morphologically, all fungal isolates resembled to Colletotrichum (Weir et al. 2012; Jayawardena et al. 2021). The internal transcribed spacer (ITS) region of the ribosomal DNA, actin (act), β-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primer pairs ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/T22 (O'Donnell and Cigelnik 1997), CL1C/CL2C (Weir et al. 2012), and GDF1/GDR1 (Templeton et al. 1992), respectively. The ITS (ON740892 to ON740894), act (ON759242 to ON759244), tub2 (ON759245 to ON759247), CAL (ON759248 to ON759250), and GAPDH (ON759251 to ON759253) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, GAPDH, CAL, act, and tub2) maximum phylogenetic analyses indicated that all fungal isolates clustered with C. siamense ICMP 18578 (type strain) with strong statistical (99% ML) support. For pathogenicity test, asymptomatic leaves, stems and fruits detached from healthy plants were surface disinfected using 0.1% NaClO for 3 min, washed three times with sterile distilled water, and air-dried. A uniform wound (3 pores, 1 mm in width) was made at the equator of each leaf, stem and fruit using aseptic needles. Mycelial plugs (5 mm in diameter) and conidia suspensions (1 × 106 conidia/ml) of each fungal isolate grown on PDA at 25 ºC for one week were used to inoculate both wounded and unwounded samples by the detached method (Huda‑Shakirah et al. 2022; Suwannarach et al. 2022). Plugs of PDA and sterile distilled water were used as controls. Ten replications were performed for each treatment and the experiment was repeated twice. All inoculated samples were incubated in a moist chamber at 25 ºC with 90% relative humidity. The disease severity index was used to evaluate the specimens (Acar et al. 2008; Ngegba et al. 2017). After one week, both wounded and unwounded leaves that inoculated with mycelial plugs and conidia suspensions showed brown leaf spots and a weak infection. Mycelial plugs inoculated on both wounded and unwounded fruits revealed a moderate infection, but inoculation of conidia suspensions showed a weak infection. No symptoms of disease were observed on the inoculated stems. Control leaves, stems and fruits remained asymptomatic. The pathogen C. siamense was re-isolated from spot and rot lesions on PDA in order to fulfill Koch's postulates. Phoulivong et al. (2012) reported that C. siamense is a causal agent of fruit rot in rose apples cultivated in Lao and Thailand. To our knowledge, this is the first report of C. siamense causing leaf spots on rose apple plants in Thailand. Importantly, these findings will provide crucial information for epidemiologic studies and in the development of appropriate management strategies for this newly emerging disease.

Detection of brown spot and leaf blight in rice with the You Only Look Once (YOLO) algorithm

Rice is a plant that produces rice as the staple food for almost all Indonesians. Rice plays a vital role in the food chain in Indonesia. However, many types of diseases on rice leaves attack and inhibit the growth of rice in Indonesian rice fields. Among them are brown spots and leaf blight. So far, the classification of diseases on rice leaves is only based on the experience of farmers. This results in uncertainty and diagnostic inaccuracy in classifying various diseases on rice plants. In this case, a method is needed to solve the problem. The method used in this research is digital imagery by processing images of diseases on the leaves of rice plants using the YOLO (You Only Look Once) algorithm. YOLO uses a single Convolutional Neural Network (CNN) for object classification and localization using Bounding Box. This research aims to provide options to users in determining the diagnosis of leaf spots and blight diseases on the leaves of rice plants. The average Precision (mAP) evaluation result is 69%, indicating that this method is suitable for detecting diseases on rice leaves.

Registration of ‘L 14‐267’ sugarcane

Abstract‘L 14‐267’ (Reg. no. CV‐213, PI 701487) sugarcane (Saccharum spp. hybrid) was released for commercial production in Louisiana because of its high yield potential and broad‐spectrum disease resistance. The final testing stage resulted in data from 62 trials at 12 representative sandy silt (light‐textured) and clayey (heavy‐textured) soil locations. Cane yield of L 14‐267 was comparable to that of ‘L 01‐299’, the most widely grown cultivar in the Louisiana sugar industry, in the plant and first ratoon crops but significantly lower in the second ratoon crop. Sucrose content was significantly higher in L 14‐267 than in L 01‐299 in the plant and first ratoon crops and was comparable to that of L 01‐299 in the second ratoon crop. Sugar yield was significantly higher in L 14‐267 than in L 01‐299 in the plant crop, comparable in the first ratoon crop but significantly lower in the second ratoon crop. The performance of L 14‐267 did not differ significantly between the two soil types for any of the traits. L 14‐267 is a mid‐ to late‐season maturing cultivar that appears to respond well to plant growth regulators but poorly to sub‐freezing temperatures. L 14‐267 displayed broad‐spectrum disease resistance, being resistant to mosaic, yellow leaf, smut, and ratoon stunt and moderately resistant to brown rust and leaf scald. L 14‐267 is moderately susceptible to the sugarcane borer and the Mexican rice borer.

Locomotor characteristics of the ground-walking chameleon Brookesia superciliaris.

Understanding the locomotor characteristics of early diverging ground-walking chameleons (members of the genera Brookesia, Rhampholeon, Palleon, and Rieppeleon) can help to explain how their unique morphology is adapted to fit their environment and mode of life. However, nearly all quantitative studies of chameleon locomotion thus far have focused on the larger "true arboreal" chameleons. We investigated kinematics and spatiotemporal gait characteristics of the Brown Leaf Chameleon (Brookesia superciliaris) on different substrates and compared them with true arboreal chameleons, nonchameleon lizards, and other small arboreal animals. Brookesia exhibits a combination of locomotor traits, some of which are traditionally arboreal, others more terrestrial, and a few that are very unusual. Like other chameleons, Brookesia moved more slowly on narrow dowels than on broad planks (simulating arboreal and terrestrial substrates, respectively), and its speed was primarily regulated by stride frequency rather than stride length. While Brookesia exhibits the traditionally arboreal trait of a high degree of humeral protraction at the beginning of stance, unlike most arboreal tetrapods, it uses smaller shoulder and hip excursions on narrower substrates, possibly reflecting its more terrestrial habits. When moving at very slow speeds, Brookesia often adopts an unusual footfall pattern, lateral-sequence lateral-couplets. Because Brookesia is a member of one of the earliest-diverging groups of chameleons, its locomotion may provide a good model for an intermediate stage in the evolution of arboreal chameleons. Thus, the transition to a fully arboreal way of life in "true arboreal" chameleons may have involved changes in spatiotemporal and kinematic characteristics as well as morphology.

A diagnosis model of soybean leaf diseases based on improved residual neural network

To solve the problems of high complexity and large computation of current disease diagnosis models based on deep learning, which are difficult to be applied to commonly used portable mobile terminals, a lightweight soybean disease diagnosis model based on attention mechanism and residual neural network was proposed. The residual attention layer (RAL) was constructed by using the attention mechanism, continuous convolution layer and shortcut connection on the traditional residual neural network, and was inserted into the residual neural network (ResNet18) in order to replace the residual structural layer. And a novel residual attention network model (RANet18) was built for soybean disease diagnosis. Based on 4301 images of soybean brown leaf spot, soybean frogeye leaf spot and soybean phyllosticta leaf spot, the RANet18 was used to conduct simulation experiments. The test result showed that the recognition accuracy was 96.50%, while the recognition time was 0.047184s, and the model size was only 40.64 MB with the F1 value was 96.43. To further verify the performance of the RANet18 on the above small sample data set, a comparative test was conducted between RANet18 and the original model, and the training time of RANet18 was 49.76% less than that of ResNet18. The model size was simplified by 4.94% and the average recognition time was saved by 45.60%. This achievement constructed a fast, efficient and accurate lightweight disease recognition model suitable for small sample data sets, and provided relevant basis and guarantee for rapid diagnosis and accurate control of crop diseases.

Recognition method of soybean leaf diseases using residual neural network based on transfer learning

Leaf disease is an important factor restricting the high quality and high yield of the soybean plant. Insufficient control of soybean diseases will destroy the local ecological environment and break the stability of the food chain. To overcome the low accuracy in recognizing soybean leaf diseases using traditional deep learning models and complexity in chemical analysis operations, in this study, a recognition model of soybean leaf diseases was proposed based on an improved deep learning model. First, four types of soybean diseases (Septoria Glycines Hemmi, Soybean Brown Leaf Spot, Soybean Frogeye Leaf Spot, and Soybean Phyllosticta Leaf Spot) were taken as research objects. Second, image preprocessing and data expansion of original images were carried out using image registration, image segmentation, region calibration and data enhancement. The data set containing 53, 250 samples was randomly divided into the training set, verification set, and test set according to the ratio of 7:2:1. Third, the convolution layer weight of the pre-training model based on the ImageNet open data set was transferred to the convolution layer of the ResNet18 model to reconstruct the global average pooling layer and the fully connected layer for constructing recognition model of TRNet18 model. Finally, the recognition accuracy of the four leaf diseases reached 99.53%, the Macro-F1 was 99.54%, and the average recognition time was 0.047184 s. Compared with AlexNet, ResNet18, ResNet50, and TRNet50 models, the recognition accuracy and Macro-F1 of the TRNet18 model were improved by 6.03% and 5.99% respectively, and the model recognition time was saved by 16.67%, The results showed that the proposed TRNet18 model had higher classification accuracy and stronger robustness, which can not only provide a reference for accurate recognition of other crop diseases, but also be transplanted to the mobile terminal for recognition of crop leaf diseases.

Characterisation and in-vitro Control of Curvularia lunata , the Causal Agent of Brown Leaf Spot Disease of Rice in Ghana

Irrigated rice production is common in the coastal savannah zone of Ghana. Recent observations have shown the incidence of a new leaf spot disease in some rice basins in the area. Research was carried out to determine the incidence and severity of the disease in the area, identify the causal agent using its cultural and morphological features, complemented with phylogenetic studies of the internal transcribed spacer region. Also, the effect of some selected fungicides and an antagonistic Trichoderma harzianum on the mycelial growth of the fungus was determined in vitro. The disease was found in all the major rice growing districts in the coastal savannah zone with severities ranging from 22.6 to 50.9%. Curvularia lunata was identified as the causal agent of the disease. Carbendazim, Nordox and Mancozeb, totally depressed the mycelial growth of the fungus whiles T. harzianum inhibited the growth of the fungus. These fungicides and antagonistic organism have the potential for the control of the disease in the field.

The Influence of the Multi-Component Mineral-Organic Concentrate on the Bonitation Value of Turfgrass

Multi-component fertilization has been found to have effects on grass metabolism, such as the stimulation of life processes and the reduction of adverse environmental conditions and pathogens. The research aimed to determine the bonitation value (assessment of the value in use) of turfgrass under the influence of using a multi-ingredient fertilizer. The experiment was carried out at the Experimental Station of the University of Agriculture in Krakow (Poland). The solution was applied through foliar application at three rates: 1.0, 2.0, and 3.0 L·ha−1. This fertilizer contains essential minerals and growth stimulants. An increase in the concentration of the test fertilizer used for spraying was associated with increased effectiveness. The plants with the highest dose of the multi-component fertilizer (treatment III) were characterized by the highest aesthetic values. The use of the concentrate reduced the occurrence of fungal plant diseases. Compared to control plants, 13% less snow mold infection and 25% fewer brown leaf spots were found. Satisfactory effects were also obtained on objects where mineral-organic concentrate was applied at a dose of 2.0 L·ha−1 (treatment II). Plants that received Treatment II and III resulted in 9% less snow mold and 15% less brown leaf spot compared to controls. In the object with the highest concentration dose (treatment III), the green index (NDVI) was also higher by 8% and the leaf greenness index (SPAD) by 7% compared to the plants from the control objects.

Brown leaf spot of Prunus serrulata Caused by Colletotrichum fioriniae in Sichuan, China.

Prunus serrulate Lindl is widely cultivated in urban areas of China. It is mainly used for wood cultivation and urban landscaping. In May 2021, new leaf spot disease was observed in Chengdu city (30°42' to 30°45'N, 103°51' to 104°7'E), with 69.3% disease incidence, which could inhibit leaf growth and reduce their biomass. A planting area of more than 1000 square meters was investigated. The diseased leaves were mostly concentrated in the lower position of plants, where the humidity was higher. The disease infected P. serrulata leaves and occurred in the field from March to October, with the highest incidence in early May. The typical symptoms initially appeared as brown necrotic lesions on the margin of the leaves. The lesions then enlarged gradually and developed into reddish brown spots, eventually coalescing into large irregular, necrotic lesions with dark brown margins. Finally, the diseased leaves withered and died. Conidiomata were not formed on the diseased tissue. Ten symptomatic leaves were collected from 5 different trees in the planting area. Infected tissues from ten samples were cut into small pieces of 3 × 3 mm. The infected tissues were surface-sterilized by 3% sodium hypochlorite and 75% ethanol respectively for 30s and 60s, and rinsed three times in sterile water. Then they were blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL), and incubated at 25°C for 4 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 67.5 to 78.6 mm. The colonies were initially white, cottony, then became light pink to misty rose at the center, and the reverse side of the colony turned dark red to red and had pale yellowish borders. The conidia were straight, smooth-walled, colorless, fusiform with acute ends, measuring 8.2 to 16.7 × 3.1 to 5.9 μm in size (n = 100). For molecular identification, the genomic DNA of the representative isolate RBWY202105 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], histone3 (HIS3) [CYLH3F/CYLH3R (Crous et al. 2004)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], actin (ACT) [ACT512F/ACT (Carbone & Kohn, 1999)], β-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: ON000436, HIS3: ON014581, CHS-1: ON014579, ACT: ON014583, TUB2: ON014582, and GAPDH: ON014580). BLAST results indicated that the ITS, HIS3, CHS-1, ACT, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P sequences at NCBI (GenBank MW497230 (561/582), MT740312 (415/415), KU736865 (258/258), MK680659 (246/246), MK967342 (757/757), and MW656269 (263/263)). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 4-year-old P. serrulata plants for pathogenicity test. Fifteen leaves of each plant were inoculated with spore suspensions on the leaves (600 μl per leaf). The same amount of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under a 16 h/8 h photoperiod and 67 to 78% relative humidity. After ten days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum fioriniae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis (The ITS, HIS3, TUB2, CHS-1, GAPDH and ACT genes). The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on P. serrulata caused by C. fioriniae in China. The identification of C. fioriniae could provide relevant information for taking management strategies and further research on the Prunus serrulata disease.

First Report of Brown Leaf Spot of Elymus nutans Caused by Bipolaris sorokiniana in Northwestern China.

Elymus nutans is an important feed and forage resource in alpine region of northwestern China, and helps to maintain the ecological balance on Qinghai-Tibetan plateau (Fei et al. 2010). In September 2021, brown leaf spots were observed in Shandan Horse Farm (38.3061°N, 101.2453°E), Gansu province of China.Disease severity of leaves was estimated to be 38% based on a random sample of 100 leaves from each of 10 arbitrarily placed quadrats on the farm.The typical symptoms on leaves appeared as brown to dark brown spots with grayish white in the center, surrounded by a yellow halo. A total of twenty diseased leaves were collected from five different plants in the field. The lesions (about 5 mm) from these samples were initially soaked in 75% ethanol for 30 s followed by soaking in 1% NaClO solution for 1 min, repeatedly rinsed with sterilized water, air dried, and put on potato dextrose agar (PDA). After 7 days of incubation at 25°C in the dark, the same fungus was consistently isolated; the pure isolates were obtained by single-spore cultures (Cai et al. 2009). The colonies were irregular in shape and black in color with white margins. Conidia were spindle-shaped, dark brown, 5 to 9 septa (7 septa in general), and a size range of 21.74 to 78.97 × 7.95 to 21.12 µm (avg. 57.25 × 17.76 µm, n = 50),morphologically similar to B. sorokiniana reported on Avena nuda in China (Li et al. 2019). The ITS region of rDNA and partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of the representative isolate PJC5 were amplified using the primers ITS1/ITS4 and gpd1/gpd2, respectively (Manamgoda et al. 2014). After sequencing sequences were deposited into GenBank with accession numbers OM419194 (ITS) and OM718009 (GAPDH). BLAST analysis showed 99% and 100% identity with the corresponding sequences (accession numbers MH855159.1 and HG779088.1) of known strain CBS 140.31 of B. sorokiniana (Manamgoda et al. 2014). For pathogenicity tests, ten 8-week-old healthy plants obtained by growing E. nutans in pots were spray-inoculated separately with a conidia suspension of 1 × 106 conidia/ml of isolate PJC5. Ten plants were also sprayed with sterilized distilled water as controls. Then all plants were individually covered with transparent polyethylene bags for three days to maintain high relative humidity and placed in greenhouse at 25°C. Seven days after inoculation, symptoms of leaf infection were similar to those observed in the field, while no symptoms appeared on leaves of control plants. The experiments were conducted three times and the pathogen was re-isolated from inoculated leaves and was confirmed as B. sorokiniana based on morphological and molecular analyses.B. sorokiniana has been reported to cause leaf spot of E. riparius in the United States (Roane et al. 2005) and E. breviaristatus (Zhuang, 2005) and A. nuda (Li et al. 2019) in China. To our knowledge, this is thefirstreportofB.sorokinianacausing leaf spot onE. nutansin China. Identifying this pathogen provides a foundation to prevent this complex disease and to reduce economic loss.

First Report of Brown Leaf Spot Caused by Neofusicoccum parvum on Photinia × fraseri Dress (Christmas berry) in China.

Christmas berry (Photinia × fraseri Dress), known as red tip photinia, is an important landscape color plant in South China (Zhu et al., 2021). In summer 2021, brown leaf spots of Christmas berry were observed with an incidence of 45-70% on the campus of Nanchang Institute of Technology, Jiangxi Province (28°41'32.61"N, 116°1'53.75"E), China. These spots have a negative influence on the photosynthetic activity and severely reduce the ornamental value of these plants. Lesions originally occurred on the margins or tips of the leaves as red patches (Figure 1 A) and then developed into irregular reddish-brown necrotic spots with yellow halos (Figure 1 B) that finally were grey lesions with dark brown ring circles surround with yellow halos (Figure 1 C). Ten leaf samples with typical symptoms from five plants were collected. The tissue between the healthy and necrotic area (ca. 5 mm × 5 mm) was cut with a sterile scalpel and surface sterilized in 70% alcohol for 45 s, followed by 2% NaClO for 2 min and washed in sterile deionized water three times, The sterilized leaf parts were placed on potato dextrose agar (PDA) and incubated at 25℃ for 3 to 5 days. A total of 28 fungal isolates were obtained from ten symptomatic leaves, which were classified into five species by morphological characteristics. Neofusicoccum was the genus with the highest isolation frequency (50%). For pathogenicity tests, healthy leaves of Christmas berry were surface-sterilized, wounded using a sterile needle and inoculated with mycelial plugs (6 mm in diameter) on the left site of the leaves. Five representative isolates were chosen and ten leaves were used for every isolate. The controls on the same leaves on the right side of the leaves were treated with sterile PDA plugs. All samples were placed in an artificial climate box (RH 90±3%, 27±1℃, 12 h light) for 3 days. Only the leaves inoculated with isolate NH7 showed dark brown leaf spots (Figure 1 D). The control leaves and those inoculated with the other four isolates remained symptomless. To confirm pathogenicity, mycelial plugs of isolate NH7 were applied on punctured leaves of Christmas berry in the field as the artificial climate box (Figure 1 E-H). Inoculation with a sterile PDA plug served as control. All the leaves were covered with plastic bags for 48 h to maintain high relative humidity. Seven days later, symptoms similar to those observed in the field developed on all leaves inoculated with isolated NH7, while the controls remained symptomless. To fulfill Koch's postulates, fungal isolates were reisolated from symptomatic leaves and identified by morphological and molecular characteristics. The colony of isolate NH7 developed aerial hyphae, which had a grey center surrounded by grey-white hyphae (Figure 1 I). Conidia were aseptate, primarily fusiform and measured 11.1-19.8 × 3.2-7.1 µm (n = 50) (Figure 1 J). For further confirmation of the identity, five genes, including ITS (White et al., 1990), RPB2 (Pavlic et al., 2009), tub2 (Glass & Donaldson, 1995), and tef1 (Carbone & Kohn, 1999) were sequenced. The sequences were deposited in GenBank (OL584411 for ITS, OL606622 for ACT, OL694623 for tub2, OM141481 for RPB2 and OM141482 for tef1), Based on the phylogenetic tree analysis using IQ-TREE, isolate NH7 clustered with Neofusicoccum parvum (Figure 2). N. parvum has been reported to cause leaf spot disease on different plants including Myristica fragrans (Jayakumar et al., 2011), Ginkgo biloba (Mirhosseini et al., 2014), Vitis heyneana (Wu et al., 2015), rubber tree (Liu et al., 2017) and Geodorum eulophioides (Du et al., 2021). To our knowledge, this is the first report of N. parvum causing leaf spot disease on Christmas berry in China. As one of the most widely planted ornamental shrubs in China, the detailed knowledge of the pathogens targeting Christmas berry is critical.

Effectiveness of fungicidal protection of spring wheat against powdery mildew and tan spot

The results of the study of the effectiveness of plant protection products against powdery mildew (Blumeria graminis) and tan spot (Pyrenophora tritici-repentis) for spring wheat are presented. It has been noted that the probability of mass destruction of crops by phytopathogens is determined by the stock of the infectious beginning of pathogens, the susceptibility of the host plant and the favorable weather conditions for their development and spread. The experiment was carried out using the methods generally accepted in the Russian Federation. During the observation period, the degree of plant damage of more than 20% in the heading phase was noted in 46% of the years of observation (in 2009, 2013, 2014, 2016, 2017). Epiphytotic development of powdery mildew together with brown leaf rust (Puccinia triticina Eriks.) was registered in 2013–2014 and 2016–2017. For 2009–2019 the degree of plant damage of more than 20% in the heading phase was noted in 46% of the years of observation (in 2009, 2013, 2014, 2016, 2017). Effective protection of wheat against powdery mildew (75–87%) was provided by fungicides based on 2–3 active ingredients. The biological effectiveness of the biofungicide (Bacillus subtilis) was manifested only in the years with moderate wheat damage and did not exceed the average level of 58%. It is more expedient to carry out operational control of yellow spotting of wheat leaves with preparations based on such active substances as "azoxystrobin + epoxiconazole", "tebuconazole + propiconazole". The level of statistically significant yield preserved due to fungicidal protection during the years of depression was 5–6%, with a moderate development of leaf diseases due to chemical fungicides - 18%, and the biological product provided an increase in productivity of 9%. During the years of epiphytoties, the chemical protection of crops retained an average of 24% of the wheat yield, polycomponent preparations had the best performance and stability of action, and the biological protection of plants under these conditions was ineffective.

First Report of Leaf Spot Caused by Alternaria tenuissima on Panicle Hydrangea (Hydrangea paniculata) in China.

Panicle hydrangea (Hydrangea paniculata), belonging to the Saxifragaceae family (Wu et al. 2001), is an ornamental flowering plant which is native to China and Japan. In July 2021, brown leaf spots (diameter ranged from 3 to 5 mm) were observed on panicle hydrangea plants in a 0.1 ha field of Northeast Agriculture University (126.72°E, 45.74°N), Heilongjiang Province. The incidence was approximately 30%. Small circular or irregular brown spots initially appeared on the older leaves, and these lesions gradually expanded with time. In some serious cases, lesions joint together and caused leaf wilting. To identify the causal agent, ten symptomatic leaves from different panicle hydrangea plants were collected, and surface sterilized with 70% ethanol for 30 s, followed by 0.5% NaClO treatment for 4 min, and then rinsed with sterile water three times. Tissues between healthy and necrotic area were cut into 5×5 mm pieces after air drying on sterile filter paper. The pieces were placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter) and incubated at 25°C for 5 days. Fifteen pure isolates were obtained using the hyphal tip technique and cultured on PDA for 7 days at 25°C for morphological and molecular identification. Colonies of all isolates were dark olivaceous with white margin. Conidiophores were septate, singly arising, and light brown, with a size range of 10.2-60.1 × 1.4-5.6 µm (n=50). Conidia were obclavate to obpyriform, brown to dark brown, and in a size range of 15.0-25.2 × 5.1-15.3 µm (n=50). To further identify these isolates, four different genomic DNA regions including ribosomal DNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (rpb2), and histone 3 (HIS3) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F2/RPB2-7R (Sung et al. 2007), and H3-1a/H3-1b (Xu et al. 2022) for three representative isolates (DXQ2-2, DXQ2-3, and DXQ2-4), respectively. The sequences of ITS, GADPH, rpb2, and HIS3 for these three isolates were identical and only sequences of DXQ2-2 were deposited in GenBank (GenBank accession nos. OL305828, OL333601, OL333602, and OL436242). These sequences showed 100%, 100%, 100%, and 99.61% identity with A. tenuissima BJ-CX-1 (GenBank accession nos. MK683974, MK683784, MK684069, and MK683879), respectively. Based on morphological features and DNA sequences analyses, these isolates were identified as Alternaria tenuissima (Simmons 2007). To fulfill Koch's postulates, ten healthy and surface disinfected leaves of one panicle hydrangea plant grown in pot were sprayed with a conidial suspension (1×106 conidia/mL) of isolate DXQ2-2. Meanwhile, ten surface disinfected leaves of another panicle hydrangea plant grown in pot sprayed with sterilize water served as the control. All plants were maintained in a greenhouse at 25℃ and 70% relative humidity. Five days after inoculation, leaves inoculated with conidial suspension showed leaf spot symptoms that were similar to those observed in the field, whereas no symptom was observed on the control leaves. The experiment was conducted twice. The Alternaria tenuissima isolate was successfully re-isolated from the symptomatic leaves and confirmed based on above morphological and molecular methods. To our knowledge, this is the first report of leaf spot on panicle hydrangea caused by A. tenuissima. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, so further investigation and management is needed to control this disease.

First report of Neofusicoccum parvum causing leaf spot disease on Macadamia integrifolia in China.

The macadamia industry is developing rapidly in China. A brown leaf spot disease was noted in six Macadamia integrifolia plantations in Lincang, Yunnan, in October 2021. Over 60% of trees sampled had brown leaf spot symptoms, among approx.15,000 trees planted in these areas. Lesions (3 to 5 mm dia.) were small round brown spots with yellow edges. Lesions on severely infected leaves were darker and larger, with irregular shape (8 to 10 mm long, 3 to 6 mm wide). About 10% of diseased leaves had lesions characterized by a shot hole surrounded by a yellow halo. Potential pathogens were isolated from four randomly-selected symptomatic leaves from each of the six plantations by cutting lesion edges into small pieces. The pieces were surface sterilized, placed onto water agar containing 100 ppm aureomycin and incubated for 5 days at 24°C in the dark. Subculturing microbial growth on potato dextrose agar produced single-hyphal isolates with white fluffy aerial mycelia that turned pale olivaceous gray after 4 to 5 days. In four randomly-selected cultures, conidia were single celled, hyaline, spindle shaped to oval, and measured 10.9 to 16.3 µm long and 4.0 to 6.2 µm wide (n = 50). These characteristics matched those of Neofusicoccum parvum (Pavlic et al. 2009). Isolate LC013 was randomly selected as a representative individual for molecular identification. Internal transcribed spacer (ITS; ITS1/ITS4 primers; White et al. 1990), beta-tubulin gene (tub2; BT2A/BT2B primers; Glass and Donaldson 1995) and translation elongation factor 1-alpha gene (tef1-α; EF1-728F/EF2 primers; Carbone and Kohn 1999; O'Donnell et al. 1998) regions were PCR amplified from genomic DNA. Sequences of the products were used to BLAST probe the type specimen nucleotide sequences in GenBank. The LC013 sequences (GenBank accessions OM392021 (ITS); OM453641 (tub2); OM567656 (tef1-α)) had >99% sequence identity with analogous sequences from the type specimen of N. parvum CBS 138823 (accessions AY236943 (ITS); AY236917 (tub2); AY236888 (tef1-α)). Isolate LC013 was sister to N. parvum type strain in a maximum-likelihood (ML) tree constructed from analogous concatenated ITS, tef1-α, and tub2 sequences of 27 species that are phylogenetically closely-related to LC013 based on the ITS single locus ML tree. Koch's postulates were tested twice with two isolates by wounding leaves of four 14-month-old M. integrifolia seedlings with a sterile needle and placing a 5-mm-diameter agar plug containing N. parvum on the wound site. PDA plugs alone were used as uninoculated controls. Leaves were covered with sealed bags to maintain >90% humidity for 24 hours. All plants were kept in the same glasshouse under natural conditions. Leaves of inoculated plants began to discolor at 5 days post-inoculation (dpi). Brown spot symptoms were observed at 9 dpi. Control plants were symptomless. N. parvum was re-isolated from leaf lesions of the infected plants, but not from control plants, thus fulfilling Koch's postulates. N. parvum is an aggressive pathogen that causes severe disease on important tree and woody species, including M. integrifolia (Liddle et al. 2019). In China, it has been reported to cause leaf spot disease on 26 plant species (Farr and Rossman 2022), but this is the first report of N. parvum causing leaf spot disease on M. integrifolia. Further investigation is required to estimate the importance of this pathogen to the macadamia industry in China.

Diagnosis and Control of Brown Leaf Spot of Kiwi (Actinidia deliciosa) Using Biochar-Zinc Oxide Nanocomposite (MB-ZnO) as a Non-Toxic Bio-Fungicides

Kiwi is one of the best natural sources of vitamin C and has wide applications. During October–November 2021, small brown spots were examined on the Kiwi leaves. The diseased leaf samples were collected and placed on potato dextrose agar nutrient media for diagnosis. Morphological, anatomical, and molecular studies revealed this disease-causing agent to be Rhizopus oryzae. Molecular characterizations of the isolated pathogen were performed by using actin translation elongation factor (EF-1α) and ribosomal deoxyribose nucleotide inter transcribed sequence (rDNA ITS ITS1/ITS4) and elongation factors (EFl-F/EFl-R) primers. A BLAST study of the resultant ITS1/ITS4 sequence showed > 99% resemblance with R. oryzae (MT603964.1), while the EF-1α sequence revealed 100% similarity with translation elongation factor-1α gene of R. oryzae (MK510718.1). The obtained ITS1/ITS4 sequence was submitted to NCBI (MW603842.1). Koch’s postulates established the pathogenicity of isolated R. oryzae and proved it to be the brown spot pathogen of Kiwi. For the environmentally-friendly management of Kiwi leaf spot, maize biochar-Zinc Oxide (MB-ZnO) nanocomposite was used. The prepared nanocomposite was characterized by Fourier transform infrared (FTIR) spectroscopy, thermo gravitational analysis (TGA), X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) analysis. After successful preparation, MB-ZnO was assessed for its possible antifungal potential against R. oryzae. MB-ZnO displayed substantial growth inhibition, and the highest growth inhibition (79%) was observed at a 19 mg/mL dose rate of nanoparticles. These excellent findings propose that Ball-milled synthesis is a fast, economical, and environmentally friendly method for nanocomposite in the near future. The nanocomposite is used as a nominal substitute for chemical fungicides.

First report of Paramyrothecium roridum causing leaf spot and stem rot diseases on Coffea arabica in Peru.

Peru is the ninth exporter of coffee (Coffea arabica) in the world, and Amazonas is among its most important producing departments (INIA 2019). In July 2021, in the nursery of the "Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva", in Huambo district (6° 26' 11.19'' S; 77° 31' 24.18'' W), four-month-old coffee seedlings cv. Catimor with 0.5-2.0 cm brown concentric leaf spots and rotten stems, bearing white mycelial tufts and black sporodochia, were observed at 30% incidence. Infected seedlings were collected. Foliar sections of 2-3 mm with infected tissue were surfaced disinfected in 2% NaClO and transferred onto Petri plates containing potato dextrose agar medium (PDA). The plates were incubated at 25° C for 7 days. We obtained three isolates (INDES-AFHP61, INDES-AFHP62 and INDES-AFHP66) with similar morphology from different seedlings. Colonies (16-17 mm diam.) formed concentric rings with white aerial mycelium, giving rise to viscous and olivaceous dark green sporodochial conidiomata. Conidia (4.82-5.77 × 1.34-1.65 µm; n = 30) were cylindric, hyaline, smooth, and aseptate. These morphological features correspond to Paramyrothecium spp. (Lombard et al. 2016). The DNA of isolates was extracted using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin), and the internal transcribed spacer 1 and 2 intervening the 5.8S subunit rDNA region (Accession numbers: OM892830 to OM892832), and part of the second-largest subunit of the RNA polymerase II, the calmodulin and the β-tubulin genes (OM919453 to OM919461) were sequenced following Lombard et al. (2016). All sequences had a percent identity greater than or equal to 99% to corresponding sequences of the P. roridum type specimen (CBS 357.89). Additionally, a multilocus Maximum Likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al. 2016; Pinruan et al. 2022) grouped our three isolates together with the type and other specimens of P. roridum in a strongly supported clade, confirming the species identification. To evaluate pathogenicity, four-month-old coffee seedlings cv. Catimor were sprayed with 10 mL of conidial suspensions at 1 x 106 /mL. A set of control seedlings were inoculated with sterile water. Seedlings were maintained in a humidity chamber at 25 °C. After 15 days brown concentric foliar spots, stem rotting, mycelial tufts and sporodochia (same symptoms and signs observed originally at the nursery) arose in the non-control seedlings. The pathogen was re-isolated on PDA, confirming P. roridum was the causal agent of leaf spot and stem rot diseases of coffee. Paramyrothecium roridum has wide geographic distribution and host range (Lombard et al. 2016). This pathogen was reported to infect C. arabica in Mexico and Coffea sp. in Colombia (Pelayo-Sánchez et al. 2017; Lombard et al. 2016; Huaman et al. 2021). It was also reported in Africa infecting soybeans (Haudenshield et al. 2018), in Brazil infecting Tectona grandis (Borges et al. 2018), in Egypt infecting strawberries (Soliman 2020), and in Malaysia infecting Eichhornia crassipes (Hassan et al. 2021). To the best of our knowledge, this is the first time P. roridum is reported on coffee in Peru.