First Report of Neofusicoccum parvum Causing Leaf Spot on coral trees (Viburnum odoratissimum) in China.

Coral trees (Viburnum odoratissimum), as a class of evergreen shrubs, are mainly planted in landscapes in numerous cities in China. During September 2020, the author investigated four major parks in Hefei (Bao Park, Hefei Botanical Garden, Luzhou Park and Peninsula Park) and the campus of Anhui Agricultural University (approximately 0.5 ha) (31°49'21.30″N, 117°13'18.25″E). The results showed that the incidence rate of leaf spot disease reached 60% among approximately 100,000 coral trees planted in these areas. Coral trees begin to show leaf spots in August. In early stages of coral trees infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. After the disease patches expand and darken, the coral leaves eventually wither and fall, which seriously affects its viewing and admiring value. To identify the fungal pathogen, the five-point sampling method was used to take typical similar leaf samples from 5 regions, and 6 samples were taken from each site, so a total of 150 samples were obtained. Fragments of sample leaves were surface-sterilized with 1% NaClO, plated on potato dextrose agar, and incubated at 25 °C in the dark. A total of 275 strains were obtained from 150 samples. According to the morphological characteristics, 275 strains were purified and divided into four types. Four representative strains (MI1, K1, F1, D1) were selected from four types for further pathogenicity testing and identification. The pathogenicity test was conducted in triplicate by inoculating wounded leaves of 1-year-old potted V. odoratissimum with 20µL of a conidial suspension (106 conidia/mL). The control was inoculated with sterile water. The specimens were placed in a growth chamber while maintaining 90% relative humidity and 28℃. After five days, the characteristic lesions were observed only on inoculated MI1 spore suspension leaves. The same fungus was reisolated from the lesions, thus fulfilling Koch's postulates. The pathogenic fungi accounted for 60% of all strains. Fungal colonies were circular and had abundant white aerial mycelium, and colonies changed from white to pure black after maturity. Conidia were fusiform (16-17×5-6 µm), thin-walled, transparent, and without diaphragms. Molecular identification was performed by partially sequencing the internal transcribed spacer (ITS) gene, the translation elongation Factor 1-alpha(EF1-alpha) gene, and the ß-tubulin (TUB2) gene by using the primers ITS1/ITS4 (White et al. 1990), EF1-728F (Alves et al. 2008)/EF1-986R (Carbone & Kohn 1999), and Bt2a/Bt2b (Glass & Donaldson 1995), respectively. The obtained ITS sequence (MW767713) showed 99% identity with N. parvum CMW28429 (KU997429.1), the EF1-alpha sequence (MZ398261) showed 99% identity with N. parvum isolate A4 (FJ528597.1), and the TUB2 sequence (MZ398260) showed 99% identity with N. parvum isolate BO52 (KU554657.1). By combining the sequences of individual fragments of each fungus in the order ITS, EF1-alpha and TUB2, MEGA 6.0 was used to analyze the sequence of kinship by using the maximum likelihood method, and the repeat value of bootstraps was 1000. A polygenic phylogenetic tree analysis based on multilocus alignment (ITS, EF1-alpha and TUB2) was constructed with some strains of Botryosphaeriaceae species. The results of the phylogenetic tree showed that MI1 and N. parvum clustered into a branch. To our knowledge, this is the first report of N. parvum causing leaf spot on V. odoratissimum in China.

Identification and Characterization of Colletotrichum Species Associated With Camellia sinensis Anthracnose in Anhui Province, China.

Recent advances in Colletotrichum taxonomy have led to the need to conduct fresh surveys of Colletotrichum species associated with important crops. Anthracnose caused by Colletotrichum spp. is one of the destructive diseases on Camellia sinensis. In this study, a total of 22 representative Colletotrichum isolates were obtained from diseased leaves of Ca. sinensis cultivated in four tea plantation regions in Anhui Province of China. The isolates were identified based on multilocus (ITS, ACT, CAL, CHS-1, TUB2, GAPDH) phylogenetic analyses, and their morphological characteristics were also analyzed. Twenty-one isolates belonging to C. gloeosporioides complex were identified as C. camelliae, C. fructicola, and C. siamense. One isolate belonging to C. boninense complex was identified as C. karstii. Pathogenicity tests revealed that the isolates of C. camelliae and C. fructicola were highly virulent when inoculated on the leaves of detached twigs of Ca. sinensis cv. Shuchazao. Furthermore, it was found that the interspecies virulence was less distinct and individual isolates showed varied virulence when inoculated on different varieties of Ca. sinensis. To our knowledge, this is the first report of C. fructicola, C. siamense, and C. karstii causing anthracnose on Ca. sinensis in Anhui Province, China.

Activity of A Novel Succinate Dehydrogenase Inhibitor Fungicide Pydiflumetofen Against Fusarium fujikuroi causing Rice Bakanae Disease.

New fungicides are tools to manage fungal diseases and overcome emerging resistance in fungal pathogens. In this study, a total of 121 Fusarium fujikuroi isolates were collected from various geographical regions of China and their sensitivity to a novel succinate dehydrogenase inhibitor (SDHI) fungicide 'pydiflumetofen' was evaluated. The 50% effective concentration (EC50) value of pydiflumetofen for mycelial growth suppression ranged from 0.0101 to 0.1012 µg/ml and for conidial germination inhibition ranged from 0.0051 to 0.1082 µg/ml. Pydiflumetofen-treated hyphae showed contortion and increased branching, cell membrane permeability, and glycerol content significantly. The result of electron microscope transmission indicated that pydiflumetofen damaged the mycelial cell wall and the cell membrane, and almost broke up the cells, which increased the intracellular plasma leakage. There was no cross-resistance between pydiflumetofen and the widely used fungicides such as carbendazim, prochloraz, and phenamacril. Pydiflumetofen was found safe to seeds and rice seedlings of four rice cultivars, used up to 400 µg/ml. Seed treatment significantly decreased the rate of diseased plants in the greenhouse as well as in field trials in 2017 and 2018. Pydiflumetofen showed superb results against the rice bakanae disease (RBD), when used at 10 or 20 g a.i./100 kg of treated seeds, providing over 90% control efficacy (the maximum control efficacy was up to 97%), which was significantly higher than that of 25% phenamacril (SC) at 10 g or carbendazim at 100 g. Pydiflumetofen is highly effective against F. fujikuroi growth and sporulation as well as RBD in the field.

First report of Meloidogyne graminicola on rice in Anhui province, China.

Meloidogyne graminicola, a devastating plant pathogen of rice (Oryza sativa), is considered to a severe constraint to productivity in rice-growing areas (Zhan et al., 2018). In April 2020, irrigated paddy rice field in Qianshan City, Anhui Province, China, showed symptoms with stunting, thinning, chlorosis, and typical hook-shaped root tips. Females and egg masses of Meloidogyne sp. were found inside the cortex of the root galls, males were found in soil and roots. The morphological characteristics of females, males and second-stage juveniles (J2) were identified as described previously (Tian et al., 2017; Wang et al., 2017). The perineal pattern of the females (n=12) was dorsoventrally oval with low and round dorsal arches, with lateral fields obscure or absent. Most of the striae were smooth, and occasionally broken by short and irregular striae. Morphological measurements (mean±sd, range) of female nematodes (n=20) were body length (543.0±66.0 µm, 448.0-629.0 µm), stylet (11.6±1.9 µm, 7.9-14.2 µm), dorsal pharyngeal gland orifice to stylet base (DGO) (4.0±0.4 µm, 3.4-4.7 µm), vulval slit length (24.1±4.9 µm, 14.8-32.8 µm), vulval slit to anus distance (16.1±3.0 µm, 8.4-20.2 µm). The male nematodes were cylindroid, vermiform, and rounded tail, with the measurements (n=20) body length (1673.0±125 µm, 1346.0-1822.0 µm), stylet (15.5±0.8 µm, 14.0-17.1 µm), DGO (3.7±0.5 µm, 2.9-5.5 µm), and spicule (30.7±2.5 µm, 23.4-34.6 µm). The J2 were vermiform with a gradually tapering hyalines, its measurements (n=20) were body length (452.0±33.0 µm, 391.0-511.0 µm), stylet (13.4±0.8 µm, 12.0-15.2 µm), DGO (3.6±0.6 µm, 2.5-4.7 µm), tail length (72.1±5.2 µm, 59.8-84.8 µm) and hyaline tail terminus (21.7±2.5 µm, 18.0-29.7 µm). DNA extracted from individual females (n=10) were used for molecular identification. The D2/D3 region of 28S RNA was amplified with D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') (De Ley et al. 1999). The ITS region was amplified with AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') and TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') (Curran et al. 1994). The fragments of D2/D3 region (GenBank accession No. MT576694) and ITS region (GenBank accession No. MT573412) were 766 bp and 579 bp respectively, they all exhibited 99%-100% similarity with those of M. graminicola isolates available in the GenBank. Therefore, both morphological and molecular characterization confirmed the status of this nematode as Meloidogyne graminicola. In green house test, twenty 2-week-old rice seedlings (cv. Longliangyou) were individually maintained in pots with sterilized sand and soil (3:1) and inoculated with 300 J2, other ten non-inoculated rice seedlings as negative control. Rice seedlings were grown in green house at 28℃/25℃ with a 16 h/8 h light/dark photoperiod. After 30 days, all inoculated rice seedling showed symptoms with stunting, chlorosis, and typical hook-shaped root tips, which were similar with that in fields. The nematode reproduction index (final population density/initial population density) were 7.86-10.32. No symptoms were observed on non-inoculated rice seedlings. These results confirmed the pathogenicity of M. graminicola on rice. To our knowledge, this is the first report of M. graminicola in Anhui Province, China. References Curran, J., et al. 1994. Mycol. Res. 98:547. https://doi.org/10.1016/S0953-7562(09)80478-4. De Ley, P., et al. 1999. Nematology. 1:591. https://doi.org/10.1163/156854199508559. Tian, Z., et al. 2017. Plant Disease. http://dx.doi.org/10.1094/PDIS-06-17-0832-PDN. Wang, G., et al. 2017. Plant Disease. http://dx.doi.org/10.1094/PDIS-12-16-1805-PDN. Zhan, L., et al. 2018. BMC Plant Biol. 18:50. https://doi.org/10.1186/s12870-018-1266-9.

Insights into the Draft Genome Sequence of the Kiwifruit-Associated Pathogenic Isolate Pseudomonas fluorescens AHK-1.

Pseudomonas fluorescens is a physiologically diverse species of bacteria present in many habitats, which possesses multifunctional traits that provide it with the capability to exhibit biological control activities, promote plant health or cause plant disease. Here, we present the draft genome sequence of the kiwifruit-associated pathogenic isolate AHK-1 of P. fluorescens, which was isolated from the diseased leaves of kiwifruit plants. The genome size of AHK-1 was found to be 7,035,786 bp, with a G + C content of 60.88%. It is predicted to contain a total of 6327 genes, of which 3998 were homologous to genes in the other two sequenced P. fluorescens isolates (SBW25 and GcM5-1A) and 946 were unique to AHK-1 based on comparative genomic analysis. Furthermore, we identified several candidate virulence factors in the genome of AHK-1, including the fliA gene encoding flagellar biosynthetic protein for biosynthesis, and the genes for components of type VI, III, and IV secretion systems. This genomic resource will serve as a reference for better understanding the genetics of pathogenic and non-pathogenic strains, and will help to elucidate the pathogenic mechanisms of P. fluorescens associated with plant disease.

A push-pull strategy for controlling the tea green leafhopper (Empoasca flavescens F.) using semiochemicals from Tagetes erecta and Flemingia macrophylla.

BACKGROUND: The tea green leafhopper, Empoasca flavescens is the most important pest in Chinese tea plantations. For decades its control has been executed almost exclusively through pesticide applications. A semiochemical-based 'push-pull' strategy was tested on the leafhopper in the study. RESULTS: The odors released from Tagetes erecta and Flemingia macrophylla significantly repelled and attracted leafhoppers, respectively. These volatile compounds (46 from T. erecta and 53 F. macrophylla) were identified and quantified via gas chromatography-mass spectometry (GC-MS) analysis. Y-tube olfactometer assays indicated that thymol anisole, thymol and camphor had significant repellent effects on the leafhoppers, resulting in a ternary repellent blend at a 4:3:13 ratio. Cis-3-hexen-1-ol, cis-3-hexenyl acetate, nonanal and α-farnesene were significantly attractive to the leafhoppers, making an attractant blend at a 17:4:1:1 ratio. In the field, the push-pull strategy with the repellent dispensers placed within the tea bushes and the attractant-baited sticky traps hung 15 cm above the tea plants showed a significant control efficacy, reaching 69% and 55% at two and 14 days post-treatment, respectively, similar to those in the insecticide control plots. Additionally, the leafhopper density in the push-pull intercropping plot was 63.2 leafhoppers/100 tea shoots/visit, much lower than those in the pull intercropping plot and nonintercropping plot. CONCLUSION: Application of the push-pull strategy using both synthetic repellent and attractant, or intercropping T. erecta and F. macrophylla with tea plants, can effectively reduce the leafhopper population. This approach might have great potential as an environmentally safe control strategy against the leafhopper. © 2022 Society of Chemical Industry.

[Microclimatic niche of rice sheath blight fungus].

By using the principles and methods of niche, the microclimatic niche of rice sheath blight fungus was analyzed. The results showed that the integrative effect of ecological factors light intensity, air temperature, and humidity was higher than the effect of each of the factors, and the combination of light intensity and air temperature had higher effect than the other combinations of the three factors. At the tillering, booting, heading, and waxing stages of rice, the microclimatic niche breadth of the fungus was 0.3112, 0.4012, 0.4326, and 0.7365, respectively, i.e., increased with rice growth, and the microclimatic niche occupied by the fungus was mainly with the low light intensity, low air temperature and high humidity as the prior microclimate type.

[Mineral nutrition niche of rice sheath blight fungus].

By using the principles and methods of niche, this paper studied the mineral nutrition niche of rice sheath blight fungus. The results showed that the mineral nutrition niche breadth of the fungus at rice tillering, booting, heading, and waxing stages was 0.2710, 0.3865, 0.4252 and 0.4817, respectively, i.e., increased with rice growth, but still comparatively narrower, indicating that rice sheath blight fungus only utilized smaller parts of mineral nutrients at various growth stages of rice. The fungus always gave priority to occupy the nutrition niches where Mg, Zn and Si contents were lower, suggesting that Mg, Zn and Si contents had close relations to the resistance of rice against rice sheath blight.

Mapping of a major stripe rust resistance gene in Chinese native wheat variety Chike using microsatellite markers.

Chike (accession number Su1900), a Chinese native wheat (Triticum aestivum L.) variety, is resistant to the currently prevailing physiological races of Puccinia striiformis Westend. f. sp. tritici in China. Genetic analysis indicated that resistance to the physiological race CY32 of the pathogen in the variety was controlled by one dominant gene. In this study, BSA (bulked segregant analysis) methods and SSRs (simple sequence repeats) marker polymorphic analysis are used to map the gene. The resistant and susceptible DNA bulks were prepared from the segregating F2 population of the cross between Taichung 29, a susceptible variety as maternal parent, and Chike as paternal parent. Over 400 SSR primers were screened, and five SSR markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 on the chromosome arm 1BL were found to be polymorphic between the resistant and the susceptible DNA bulks as well as their parents. Genetic linkage was tested on segregating F2 population with 200 plants, including 140 resistant and 60 susceptible plants. All the five SSR markers were linked to the stripe rust resistance gene in Chike. The genetic distances for the markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 to the target gene were 8.3 cM, 9.1 cM, 17.2 cM, 20.6 cM, and 31.6 cM, respectively. Analysis using 21 nulli-tetrasomic Chinese Spring lines further confirmed that all the five markers were located on chromosome 1B. On the basis of the above results, it is reasonable to assume that the major stripe rust resistance gene YrChk in Chike was located on the chromosome arm 1BL, and its comparison with the other stripe rust resistance genes located on 1B suggested that YrChk may be a novel gene that provides the resistance against stripe rust in Chike. Exploration and utilization of resources of disease resistance genes in native wheat varieties will be helpful both to diversify the resistance genes and to amend the situation of resistance gene simplification in the commercial wheat cultivars in China.

[Effects of fungicide on temporal and spatial niches of Rhizoctonia solani].

This paper studied the variation of the temporal and spatial niches of rice sheath blight-causing Rhizoctonia solani under spraying fungicide. The results showed that between fungicide treatments and control, the spatial niche breadth of Rhizoctonia solani was different, while its temporal niche breadth was similar. The spatial niche breadth of Rhizoctonia solani was 0.5240 when spraying fungicide at booting and full heading stages, 0.5742 at booting and milkfilling stages, and 0.8577 at tillering stage, while the control was 0.8563. Spraying fungicide had little effect on temporal niche breath. The spatial niche breath, percentage of diseased leaves and sheathes, index of disease, and control effect all suggested that spraying fungicide two times in rice growth period was better than spraying it one time. Spraying fungicide at booting and full heading stages and at booting stage and milkfilling stages had the best effects. Fungicide could not only narrow the spatial niche breadth, limit the spread of Rhizoctonia solani on top leaves which were important for the yield, but also narrow the temporal niche breadth, limit the spread of Rhizoctonia solani during the yield formation period of rice. But, spraying fungicide only changed the distribution of the fungus niches and narrowed the niches during rice yield formation period, with no changes in the whole niches of Rhizoctonia solani.

[Effects of ecological factors on infection process of Pyricularia grisea].

Rice blast is one of the main rice diseases. In order to discuss the occurrence regularity and improve the veracity of forecasting, the effects of ecological factors on infecting process, such as rice growth period, cultivar resistance, temperature, precipitation, and fertilization of nitrogen, were studied quantitatively. The results showed that adhering conidia on leaves, or ratio of infection was negatively correlative with the growth period of rice. The latent period of Pyricularia grisea was closely related to temperature, and within the range from 10 degrees C to 33 degrees C, the latent period was the shortest at 28 degrees C. The ratio of conidia adhering on leaves was also closely related to precipitation, especially the intensity and duration of precipitation in the first 5 hours after conidia contacting leaves, and there was no obvious effect after 5 hours. Under certain conditions, the ratio of expansive lesions to non-expansive lesions, the maximum area of expansive lesions, and variety resistance of rice were related to the rank of leaves.