First Report of Colletotrichum fructicola Causing Anthracnose on Camellia yuhsienensis Hu in China.

Camellia yuhsienensis Hu is an endemic species from China, where is the predominant oilseed crop due to its anthracnose resistance (Kuang 2015; J. Li et al. 2020; Nie et al. 2020). In April 2019, anthracnose symptoms were observed on C. yuhsienensis in a plantation in Youxian, Zhuzhou, Hunan Province, China (113.32°E, 26.79°N). It was detected approximately 10% anthracnose incidence in 500 two-year-old plants in a 5000 m2 cultivated area. Diseased leaves showed irregular grayish brown spots with dark brown edges and dark brown undersides. Symptomatic tissues (4 to 5 mm2) were surface-disinfected for 90 s in 75% ethanol, then rinsed twice with sterile water, and finally incubated on PDA (potato dextrose agar) at 28℃ (Jiang et al. 2018). Pure cultures were obtained by the single-spore isolation method. A total of 100 fungal isolates were obtained from 85 symptomatic leaves, from which 81 had similar colony morphology. Colonies on PDA were white, fluffy and cottony, and becoming dark gray after 5 days. The character of the reverse of the colony were similar to that of the upper of the colony, but the color was darker at the same time. The isolates produced a large number of single-celled, hyaline, straight and cylindrical conidia, with 10.35 to 17.58 length × 3.46 to 5.69 µm width (x=13.61 × 4.63 µm, n = 30). The isolates were preliminarily identified as Colletotrichum spp. according to morphological features (Weir et al. 2012). Representative isolate YX2-5-2 was used for molecular identification: internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genomic DNA regions were amplified by PCR (Weir et al. 2012). Gene sequences were deposited in GenBank (GenBank accession no. MW398863 for ACT, MW886232 for CHS-1, MW398864 for GAPDH, MW398865 for ITS). BLAST analysis revealed that DNA sequences of YX2-5-2 at the ITS, GAPDH, ACT, and CHS-1 loci showed 100%, 99.25%, 100%, and 99.33% sequence identity, respectively to their corresponding loci in strains ZH6 (GenBank accession no. MT476840.1), ICKP18B4 (LC494274.1), YN17 (MN525804.1), and ICKG4 (LC469131.1) of C. fructicola. A Maximum Likelihood phylogenetic tree based on the combined ACT, CHS-1, ITS and GAPDH sequences revealed that the representative isolate YX2-5-2 clustered with C. fructicola. In addition, the morphological features of YX2-5-2 were similar to C. fructicola which has been reported (Weir et al. 2012). Pathogenicity was tested using isolate YX2-5-2 by inoculating leaves of 2-year-old C. yuhsienensis. Four leaves of each healthy C. yuhsienensis were sprayed with a conidial suspension (105 conidial/mL) of isolate YX2-5-2, and the above steps were repeated three times. Two additional mock-inoculated control plants were sprayed with sterilized liquid potato dextrose medium. The plants were incubated in a greenhouse at 28℃ and 90% humidity with a 12 h photoperiod. Anthracnose-like symptoms were observed 5 days post-inoculation. The control plant tissues remained healthy. C. fructicola was re-isolated on PDA from lesions, and the morphological features were consistent with YX2-5-2, confirming Koch's postulates. To our knowledge, this is the first report of anthracnose of C. yuhsienensis caused by C. fructicola in China. Anthracnose of Camellia. oleifera has been reported for a long time (H. Li et al. 2016). C. yuhsienensis, as a wild relative of C. oleifera (commonly known as tea-oil tree), has been concerned about its resistance to anthracnose. Therefore, the occurrence of C. yuhsienensis anthracnose hindered the control of anthracnose tea-oil tree. This finding will lay the foundation for studying the pathogenesis of anthracnose of tea-oil tree and developing effective prevention methods. References: Jiang, S. Q., et al. 2018. Plant Dis. 102: 674. Kuang, R. 2015. Forest Pest and Disease. Li, H., et al. 2016. PLoS One 11: e0156841. Li, J., et al. 2020. Microorganisms 8: 1385. Nie, Z., et al. 2020. Mitochondrial. DNA. B. 5: 3016. Weir, B. S., et al. 2012. Stud. Mycol. 73: 115.

First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China.

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies' undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5-20.6×3.5-6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

First Report of Colletotrichum brevisporum Causing Anthracnose of Dalbergia odorifera in China.

Dalbergia odorifera T. Chen (family Fabaceae) is one of four prized species of mahogany plant in China. In June 2017, an investigation of the condition of anthracnose was carried out on apporximately 333 hectares of D. odorifera plantations in Haikou City, Hainan Province (110.19°E, 20.03°N). Approximately 40% of D. odorifera plants had disease symptoms. Lesions on leaves were brown to grayish-white containing black dots and dark-brown borders, occasionally surrounded by a yellowish-green halo. Leaf spots generally occurred along the edge of the leaf. Severely infected leaves became withered and died. Hyphal growth was recovered from symptomatic leaf tissue, surface-sterilized with a 75% ethanol solution for 30s, rinsed with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 26°C in the dark. The representative isolate JXHTC19 was recovered by transferring a hyphal tip to a fresh PDA plate to obtain a pure culture. Fungal colonies had white aerial mycelium initially, turning pale gray after 3 days. At 7 days, colonies had a cottony appearance ranging from white to dark gray with orange masses of conidia. The colony surface was slimy and aerial mycelium was sparse. Isolates displayed single-celled, cylindrical, hyaline conidia that were rounded at both ends, and were 9.7 - 16.4 µm long (avg. 13.5 µm) × 3.6 - 6.2 µm wide (vg. 4.8 µm) (n = 100). To further identify the fungus, genomic DNA was extracted from single conidial cultures of JXHTC19. The rDNA internal transcribed spacer (ITS) region, glutamine synthetase (GS) gene, partial sequence of glyceraldeyde-3-phosphate dehydrogenase-like (GAPDH) gene, actin (ACT) gene, and beta-tubulin (TUB2) gene were amplified using the primer pairs ITS4/ITS5, GS-F/GS-R, GDF1/GDR1, ACT-512F/ACT-783R, and TUB2-T1/Bt-2b (Weir et al 2012), respectively. The results showed that the ITS, GS, GAPDH, ACT and TUB2 genes of the target strain (JXHTC19) have 100%, 95%, 100%, 97% and 98% sequence homology with C. brevisporum, respectively. The sequences obtained were deposited in GenBank (MF993572, MN737615, MN737614, MG515612, and MG515615[LJ1]). All five sequences were analyzed together with representative sequences from type or ex-type specimens of the Colletotrichum genus (Yang et al. 2011, Weir et al. 2012) and a phylogenetic tree was generated via the neighbor-joining method using MEGA6. The tree placed the isolate in the same group as C. brevisporum. Thus, both morphological and molecular characteristics identified the pathogen as C. brevisporum. To verify Koch's postulates, two-year-old leaves of healthy potted D. odorifera plants (n = 6) were inoculated with a spore suspensions of JXHTC19 that contained 105 conidia/ml. Plants were sprayed with water to serve as mock-inoculated controls [LJ2](Garibaldi et al, 2020). Six plants per treatment were used in each test. The test was repeated once.Plants were incubated in moist chambers at 26°C and monitored daily for symptom development. After five days, eleven of twelve isolates [LJ3]caused lesions on all inoculated plants, whereas no symptoms developed on the mock-inoculated controls. Koch's postulates were fulfilled by reisolating the same fungus and verifying its colony and morphological characters as C. brevisporum. To our knowledge, this is the first report of this species causing anthracnose of D. odorifera in China. Corresponding measures must be adopted to manage this disease such as reducing the planting density of D. odorifera and increasing the species diversity of undergrowth vegetation. These results could help develop better monitoring and management practices for this disease.

First Report of Fusarium fujikuroi Causing Leaf spot of Dalbergia odorifera T.Chen in China.

Dalbergia odorifera T. Chen is a national second-grade protected and one of the four famous trees in China, with high medicinal and economic value. Leaf spot disease in this plant can cause the leaves to dry up, perforate or even fall off, which affects the growth and development, and also has a great influence on its products. In May 2019, the leaf spot of Dalbergia odorifera T. Chen was found and observed in Chengmai County (N19°40', E110°0'), Hainan Province, China, and the symptomatic leaves were brought back to the laboratory for research; According to our survey at that time, the incidence of the disease was between 10% and 15%. A sterile stainless-steel scalpel was used to cut the tissues at the junction of the leaf lesions and placed on a clean bench, soaked in alcohol (75 %) for 30 s, and rinsed thrice with sterile water. Then it was inserted obliquely onto lactic acid-containing potato dextrose agar (PDA) and incubated at 28 °C for 5 days. The growing prominent colonies were singled out and re-inoculated on PDA and SNA plates. Preliminary identification was based on morphological characteristics, followed by molecular identification of strains by evaluating genes for translation elongation factor-1α(TEF-1α), beta-tubulin, mitochondrial small subunit (mtSSU)( Duan et al. 2019; Cao et al.2019; Stenglein et al.2010), and histone H3 (Jacobs, et al. 2010) . Through morphological observation, the isolate was identified as Fusarium fujikuroi. At the initial stage of growth on PDA, the strain produced a large number of white hyphae, followed by pink and purple-brown hyphae in the center of the colony which spread to the surrounding area. The microspores were abundant, colorless, elliptic or clavate, without septum or at 1-2 septate, and the size was about 3.3 to 13.5 × 1.2 to 3.2 µm. After nine days of culturing on SNA medium, few, large conidia were observed, typically sickle-like, with 3-4 septa with a size of about 20 to 40.2 × 2.3 to 4.4 µm. The identity of the strains was determined by comparing the gene sequences of TEF-1α, mtSSU, beta-tubulin and histone H3 by NCBI BLAST. The results showed that TEF-1 α (MN958396), mtSSU (MN958394), ß - tubulin (MN958395), and histone H3 (MN958397) from the target strain (jxht0302) had 100% sequence homology with F. fujikuroi (GenBank, accession numbers KF604040.1, MF984420.1, XM023575231.1, and MF356523.1 respectively). Next, the infection of D. odorifera T. Chen seedlings with and without injury was studied using a fungus block, with PDA as a control. Two days after inoculation with injury, obvious lesions were observed on the leaves, which appeared at least 5 days post- inoculation without injury, with no lesions in the control group. F. fujikuroi could be re-isolated from the leaves with lesions, but not from the control group. F. fujikuroi causes Black Rot of Macleaya cordata and maize ear rot (Yull et al.2019; Duan et al. 2019). As far as we know, this is the first report of F. fujikuroi causing leaf spot disease of D. odorifera T. Chen. Given the importance of D. odorifera T. Chen products, this disease needs more attention to tackle it.

New Mycodiplosis gall midge (Diptera: Cecidomyiidae) feeding on fungal rusts (Fungi: Pucciniomycetes) that are pathogenic on cultured plants.

Larvae of a gall midge were found feeding on the fungal rust Maravalia pterocarpi (Pucciniomycetes: Pucciniales: Chaconiaceae) infesting the economically important sua tree Dalbergia tonkinensis (Fabaceae) on Hainan Island, China. The adults, pupae and larvae were collected, their morphology was studied and a segment of the Cytochrome Oxidase unit I (COI) mitochondrial gene was sequenced. The gall midge proved to be a species new to science that belongs to the genus Mycodiplosis (Diptera: Cecdiomyiidae). Comparison of the sequence to published Cecdiomyiidae sequences revealed that, despite being undescribed and unnamed, it was previously found in east and south-east Asia to feed on several rust species: Puccinia coronata (Pucciniomycetes: Pucciniales: Pucciniaceae) that develops on Lolium multiflorum (Poaceae), Puccinia sp. on Zea mays (Poaceae), Puccinia arachidis on Arachis hypogaea (Fabaceae) and Puccinia allii on Allium fistulosum (Amaryllidaceae). The new species is described and named here Mycodiplosis puccinivora Jiao, Bu Kolesik. It occurs in China, Japan, Thailand, Bangladesh and possibly Malaysia and Australia. In Hainan it has four to five generations per year.

Population Genetic Analyses of the Fungal Pathogen Colletotrichum fructicola on Tea-Oil Trees in China.

The filamentous fungus Colletotrichum fructicola is found in all five continents and is capable of causing severe diseases in a number of economically important plants such as avocado, fig, cocoa, pear, and tea-oil trees. However, almost nothing is known about its patterns of genetic variation and epidemiology on any of its host plant species. Here we analyzed 167 isolates of C. fructicola obtained from the leaves of tea-oil tree Camellia oleifera at 15 plantations in seven Chinese provinces. Multilocus sequence typing was conducted for all isolates based on DNA sequences at fragments of four genes: the internal transcribed spacers of the nuclear ribosomal RNA gene cluster (539 bp), calmodulin (633 bp), glutamine synthetase (711 bp), and glyceraldehyde-3-phosphate dehydrogenase (190 bp), yielding 3.52%, 0.63%, 8.44%, and 7.89% of single nucleotide polymorphic sites and resulting in 15, 5, 12 and 11 alleles respectively at the four gene fragments in the total sample. The combined allelic information from all four loci identified 53 multilocus genotypes with the most frequent represented by 21 isolates distributed in eight tea-oil plantations in three provinces, consistent with long-distance clonal dispersal. However, despite evidence for clonal dispersal, statistically significant genetic differentiation among geographic populations was detected. In addition, while no evidence of recombination was found within any of the four gene fragments, signatures of recombination were found among the four gene fragments in most geographic populations, consistent with sexual mating of this species in nature. Our study provides the first insights into the population genetics and epidemiology of the important plant fungal pathogen C. fructicola.

[Soil microfauna diversity among Cunninghamia lanceolata plantations based on pyrosequencing].

In order to study the function of soil microfauna and its responses to environmental changes, we used metagenome analyses of the 18S rDNA gene region to identify differences in microfauna diversity and community structure among fifteen soil samples belonging to five different Cunninghamia lanceolate plantations. The plantations were located in Youxian County, Hunan Province in central China. The trees in these plantations were of different ages (3, 13, and 26 years) and belonged to different ecological successions (first, second, and third successions). The total dataset comprised 94922 high quality sequences with an average length of 436 bp. The dominant taxonomic groups across all samples were Chordata, Annelida, Arthropoda, Nematoda, Rotifera and Platyhelminthes with each accounting for 60.8%, 24.0%, 7.4%, 3.6%, 1.5% and 1.2% of the sequences, respectively. There were significant differences in ACE index and Shannon index among the five plantations. The lowest diversity of soil microfauna was in the 13-year old plantation of the first ecological succession. The correlation analysis showed that both ACE and available potassium concentration were negatively correlated to the Chaol index. However, there were no significant correlations between the Shannon, Simpson indices and the physical-chemical properties of soil. Overall, the Jaccard's similarity coefficient was less than 0.4 among samples at each site, and significant differences were found among plantations.

[Prediction of chlorophyll content of leaves of oil camelliae after being infected with anthracnose based on Vis/NIR spectroscopy].

The prediction model of chlorophyll content of leaves in canopies of oil camelliae under disease was explored and built by analyzing the Vis/NIR spectroscopy characteristics of oil camelliae canopies after being injected with anthracnose. Through field survey of disease index (DI), chlorophyll content and spectral data of leaves in canopies surviving different severity of disease were acquired. The first order differential of spectral data combined with moving average filter was pretreated. The prediction model of BP neural network of chlorophyll content was built by extracting sensitive wave band from spectral resample data. The results showed that with the disease being aggravated, reflection peaks and valleys of spectra of oil camelliae canopies in visible-light region vanished gradually, steep red edges from red light to near infrared leveled little by little, and reflectivity of healthy oil camelliae was far larger than that of ill ones. The sensitive wave band of absorption and reflection of chlorophyll lay in the region of 84-512, 533-565, 586-606 and 672-724 nm. The correlation coefficient r and RMSE between predictive values calculated from BP neural network using sensitive wave band as input variables and observed values was 0.9921 and 0.0458 respectively. It was therefore feasible to utilize Vis/NIR spectroscopy technology to forecast the chlorophyll content of oil camelliae after being infected with anthracnose.