First Report of Cytospora ailanthicola Causing Canker on cherry blossom (Cerasus serrulata) in China.

Cherry blossom (Cerasus serrulata) is a plant with important garden applications. It is a newly introduced exotic plants in the Arar region of Xinjiang, China (40°41'18.19″N,81°43'50.55″E). In October 2022, it was discovered that about 30% of cherry blossoms had a canker disease. The leaves of the sick branches were dired, the branches themselves were damaged, with dark brown color inside. Orange-yellow conidia horns were produced in humid condition. Samples were collected from fifteen trees exhibiting notable symptoms. The diseased junctions of the infected shoots were chopped into small pieces and subjected to surface sterilization by using 70% ethanol for 30s, 1% NaClO solution for one minute, and sterile distilled water three times (Chen et al. 2016). The representative strain YINGHUA-1 was chosen for identification by molecular biology and morphology. After five days of incubation at 26℃ on PDA media, colonies of white fluffy mycelium were produced from the YINGHUA-1 strain. After 25 days of PDA culture, the production of pycnidia was first observed, circular, black. The pycnidia began to produce conidia at 30 days. The conidia was translucent without septum, with a slightly curved single cell and smooth surface. Pycnidia was spherical and flat, with a single black aperture at the top that resembles a nearly round hole, the chamber was made up of several tiny chambers separated by a shared wall, and its diameter ranges from 900-1900 µm. The size of the conidium was 3.7-6.6×1.1-1.9 µm (n=20). The intrinsic transcriptional spacer (ITS), transcriptional elongation factor (tef-1α), and ß-tubulin (tub2) gene moieties of rDNA were sequenced using ITS1/ITS4, EF1-728F/EF1-986R, and Bt2a/Bt2b primers, respectively(Zhang et al. 2014). The amplified sequences of ITS region (Accession No. OR855907), tub2 (Accession No. OR865863) and tef-1α (Accession No. OR865864) were deposited in the GenBank. BLAST searches of the sequences revealed 99.59% identity (474/476 bp) of the ITS sequence, 98.63% identity (216/219 bp) of the tef-1α sequence, and 98.55% identity of the tub2 sequence (339/344 bp) with C. ailanthicola CFCC59446 (accessions OR826163, OR832040, and OR832062, respectively.) Phylogenetic analyses were performed with Iqtree v.1.6.12 for maximum likelihood (ML). Confidence levels for the nodes were determined using 1000 replicates of bootstrapping methods. Based on phylogenetic analysis and morphological characteristics, the pathogen was identified as C. ailanthicola. The pathogenicity of C. ailanthicola was confirmed by inoculation of 1-year-old shoots (5 replicates of this experiment). After 7 days, symptoms of inner bark discoloration were visible on xylem of branches and the same fungus was re-isolated from the inoculated shoots, with no lesions on the control shoots. C. ailanthicola is only known from a single host plant, Ailanthus altissima,in China (Fan et al.2020). As far as we know, this is the first report of C. ailanthicola harmings C. serrulata in China.

Comparative Assessment of Habitat Suitability and Niche Overlap of Three Cytospora Species in China.

The plant pathogenic fungus Cytospora is notoriously known for causing woody plant canker diseases, resulting in substantial economic losses to biological forests and fruit trees worldwide. Despite their strong negative ecological impact, the existing and prospective distribution patterns of these plant pathogens in China, according to climate change, have received little attention. In this study, we chose three widely dispersed and seriously damaging species, namely, Cytospora chrysosperma, Cytospora mali, and Cytospora nivea, which are the most common species that damage the Juglans regia, Malus domestica, Eucalyptus, Pyrus sinkiangensis, Populus spp., and Salix spp. in China. We utilized ecological niche modeling to forecast their regional distribution in China under four climate change scenarios (present, SSP 126, SSP 370, and SSP 585). The results show that temperature-related climate factors limit the current distribution ranges of the three species. Currently, the three studied species are highly suitable for northeast, northwest, north, and southwest China. Under future climate scenarios, the distribution ranges of the three species are projected to increase, and the centers of the adequate distribution areas of the three species are expected to shift to high-latitude regions. The three species coexist in China, primarily in the northwest and north regions. The ecological niches of C. chrysosperma and C. nivea are more similar. The distribution range of C. mali can reach the warmer and wetter eastern region, whereas C. chrysosperma and C. nivea are primarily found in drought-prone areas with little rainfall. Our findings can help farmers and planners develop methods to avoid the spread of Cytospora spp. and calculate the costs of applying pesticides to reduce contamination and boost yields.

Prediction of Suitable Habitat Distribution of Cryptosphaeria pullmanensis in the World and China under Climate Change.

Years of outbreaks of woody canker (Cryptosphaeria pullmanensis) in the United States, Iran, and China have resulted in massive economic losses to biological forests and fruit trees. However, only limited information is available on their distribution, and their habitat requirements have not been well evaluated due to a lack of research. In recent years, scientists have utilized the MaxEnt model to estimate the effect of global temperature and specific environmental conditions on species distribution. Using occurrence and high resolution ecological data, we predicted the spatiotemporal distribution of C. pullmanensis under twelve climate change scenarios by applying the MaxEnt model. We identified climatic factors, geography, soil, and land cover that shape their distribution range and determined shifts in their habitat range. Then, we measured the suitable habitat area, the ratio of change in the area of suitable habitat, the expansion and shrinkage of maps under climate change, the direction and distance of range changes from the present to the end of the twenty-first century, and the effect of environmental variables. C. pullmanensis is mostly widespread in high-suitability regions in northwestern China, the majority of Iran, Afghanistan, and Turkey, northern Chile, southwestern Argentina, and the west coast of California in the United States. Under future climatic conditions, climate changes of varied intensities favored the expansion of suitable habitats for C. pullmanensis in China. However, appropriate land areas are diminishing globally. The trend in migration is toward latitudes and elevations that are higher. The estimated area of possible suitability shifted eastward in China. The results of the present study are valuable not only for countries such as Morocco, Spain, Chile, Turkey, Kazakhstan, etc., where the infection has not yet fully spread or been established, but also for nations where the species has been discovered. Authorities should take steps to reduce greenhouse gas emissions in order to restrict the spread of C. pullmanensis. Countries with highly appropriate locations should increase their surveillance, risk assessment, and response capabilities.

Whole genome sequence of Cryptosphaeria pullmanensis, an important pathogenic fungus potentially threatening crop and forestry production.

Many fungal members of the Diatrypaceae family are pathogenic towards plants and are widely distributed globally. Cryptosphaeria pullmanensis is a pathogenic fungus that infects populus and walnut trees, causing their death. We sequenced the genome of C. pullmanensis based on a combination of Nanopore PromethION and Illumina NovaSeq PE150 platforms, and functionally annotated the sequences using a number of open-access databases. This is the first report of the genome-scale assembly and annotation for C. pullmanensis, the first species of the genus Cryptosphaeria to be sequenced. We obtained 13 contigs with an N50 contig size of 7,095,780 bp, a GC content ratio of 43.23% and a genome size of 56.72 Mb with 10,474 putative coding genes. Comparative genomic analysis against the genomes of seven Ascomycetes fungal strains was performed. Among the seven species tested, the Eutypa lata genome displayed the highest similarity to the C. pullmanensis genome in terms of collinearity and homologous gene content. This study has provided a genetic resource that offers extensive information and a framework for future investigations into the transcriptome, proteome, and metabonome of C. pullmanensis to understand its molecular pathogenesis.

First report of Diplodia mutila causing canker and branch dieback of walnut trees in Xinjiang, China.

Walnut (Juglans regia L.) has become an important economic fruit tree in China. In April 2022, branch dieback was observed in 15-year-old walnut trees (cv Wen 185) in a commercial orchard in Aksu, Xinjiang, China (40°21'55''N, 80°1'48''E), with an incidence of 2% (4 out of 200 trees) of affected trees. The symptoms observed, included depressed and shrunken cankers, twigs and branches dieback. Cross-sections of diseased branches revealed dark-brown wedge-shaped lesions. To isolate the potential causal pathogen, four specimens were isolated from diseased branches, and small pieces taken from the edge of canker samples (0.5 × 0.5 cm), were disinfected by immersion in 75% ethanol for 30 s and 2% NaClO solution for 3 min, and rinsed three times with sterile water. The disinfected wood samples were then placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for 2-3 days. Then, we applied the mycelial tip purification method and repeated this purification process until a single colony was formed. Four pure isolates (3-1-51A, W-2-54, K-1-43A, K-1-43B) developed white to white-gray fast-growing colonies with abundant aerial mycelium after seven days at 25°C on PDA and gradually became dark olive green over subsequent growth stages. Conidia production was then induced on 2% w/v water agar containing sterilized pine needles under near-U/V light (Alves et al., 2004). The conidia were initially hyaline, thick-walled, oblong to ovoid with one septum and a size range of 19.47-24.16 × 9.78-13.51 µm (n = 40). Based on morphological characteristics these isolates were tentatively identified as Diplodia mutila (Fr.) Mont. (Alves et al., 2004).To confirm the pathogen identified, the representative isolate 3-1-51A was amplified and sequenced using specific primer pairs (ITS1/ITS4, EF1-986/EF1-728E, BT2a/BT2b) to the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB) (White et al. 1990; Carbone and Kohn 1999; O' Donnell and Cigelnik 1997), respectively. The sequences showed 100% similarity to two D. mutila strains CBS230.30 and CBS112553. Maximum likelihood analysis was performed based on a concatenated dataset (ITS + TEF1-α + TUB) gene using MEGA 11. 0 and isolate 3-1-51A formed a single clade with the reference ex-type of D. mutila. The isolate 3-1-51A was deposited into GenBank as OP006733, OP373140, and OP373139 for ITS, TEF1-α, and TUB, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate 3-1-51A on one-year-old healthy walnut branches cv. Wen 185 of walnut trees (n=5). Five twigs of healthy walnut branches were cleaned, submerged in 1% NaClO for 15 minutes and then dried. Then, a sterile hole punch (5mm in diameter) was used to create a wound in the middle of each walnut branch, and placing mycelial plugs(3 days old; 5 mm in diameter) and sealed with parafilm. An equal number of twigs inoculated with sterile agar plugs served as controls. On the 7th day after inoculation, dark brown coloration was developed on the branches with symptoms of shrinkage dryness, and dieback. D. mutila isolate was re-isolated only from the inoculated branches. In negative control twigs, lesions and re-isolated were absent, thus fulfilling Koch's postulates. D. mutila has been previously reported causing canker and branch dieback in walnut trees in Chile (Díaz et al. 2018) and California (Chen et al. 2014). Previously, D. seriata (Zhang et al. 2017), Botryosphaeria dothidea (Guo et al. 2016), Lasiodiplodia pseudotheobromae (Guo et al. 2016), Dothiorella gregaria (Liu et al. 1986) and Neofusicoccum parvum (Yu et al. 2015) have been identified on walnut trees in China. To our knowledge, this is the first report of canker and branch dieback caused by D. mutila in walnut trees in Xinjiang, China. Further studies are now required to better understand the etiology of canker and branch dieback on walnut trees from different areas in China.

First Report of Valsa nivea (Hoffm.) Fr. Causing Valsa Canker on Korla Pear (Pyrus sinkiangensis Yü) in the World.

Korla pear (Pyrus sinkiangensis Yü) is an important commercial fruit tree that originated in China (Zhou et al. 2020). In April 2020, a survey was conducted in Aksu region, Xinjiang (40°55'37"N, 80°28'42"E), China. Some Korla pear trees (>15 years old) exhibited symptoms of branch dieback and branch cankers. Cankers observed on the trunk and branches of the tree were sunken, dark ulcerative lesions sometimes exhibiting signs of stromata erumpent through the bark and exuding yellow to reddish-orange spore tendrils. Of the 180 plants surveyed, 80% were symptomatic. Thirty samples of symptomatic tissues of infected branches were taken to the laboratory. Bark and cortical wood samples containing necrotic and healthy tissue were excised with flame-sterilized scalpels, surface disinfected with 75% ethanol and 1% NaClO, placed on PDA plates, and incubated at 25°C. A total of 30 fungal isolates were obtained. Among them, 28 isolates were identified as Valsa mali var. pyri (Lu. 1992) based on morphological and molecular identification, and two isolates (ALE6T-GP21 and ALE7T-GP23) were identified as Valsa nivea (Hoffm.) Fr. Valsa nivea isolates had a fine villi form mycelium that was initially white, turned grayish-green over time and grew close to the medium surface. Cultures also contained black ostiolate pycnidia in a stroma that consisted of multiple irregular locules. Conidiophores were hyaline, occasionally branched at the bases and (15.50-)16.48-17.94(-18.50)×(1.00-)1.13-1.37(-1.50) µm (n=20). Conidiogenous cells were phialidic and subcylindrical that taper towards the apex. Conidia were hyaline, banana-like and (5.47-)6.13-6.97(-7.64)×(1.02-)1.06-1.20(-1.23) µm (n=10). The molecular characteristics are consistent with the previous description of V. nivea (Adams et al. 2006). The internal transcribed spacer (ITS), transcription elongation factor (tef-1α) and ß-tubulin (Tub2) gene were sequenced using ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b primers, respectively (Zhang et al. 2014). BLAST (Basic Local Alignment Search Tool) searches against the NCBI database revealed that the ITS sequence had 99.83% homology (ON843984.1 and ON843987.1), tef-1α gene had 99.22% homology (MH015266.1 and MH015267.1), and the Tub2 sequence had 99.57% and 100% homologies (KT934364.1 and KT934364.1) with V. nivea sequences. The amplified sequences of ITS region (OK442665 and OK442666), tef-1α (OK510871 and OK510872) and Tub2 (OK510869 and OK510870) were deposited in the GenBank. A phylogenetic analysis was performed using MEGA7 that shows 100% bootstrap support that ALE6T-GP21 and ALE7T-GP23 were V. nivea. A pathogenicity trial was conducted with isolate ALE6T-GP21 inoculated onto 1-year-old shoots of 15-year-old Korla pear trees in Alar city, Xinjiang, China. Five shoots were inoculated by making 5-mm deep wounds using a sterile scalpel then inoculating with a 50 µL conidia suspension (1×106 mL-1). Additionally, five shoots served as the negative control and were inoculated in the same way using 50 µL ddH2O. The trees were kept under ambient conditions. Inoculated branches developed symptoms 18 days post inoculation, whereas the control branches showed no symptoms. V. nivea was re-isolated from the symptomatic areas and the isolate confirmed as ALE6T-GP21 by sequence analysis. Currently, the proven hosts of V. nivea are Populus, Elaeagnus, Juglans, Malus and Salix (Adams et al. 2006; Wang et al. 2020). To our knowledge, this is the first report of pathogenic V. nivea occurring on P. sinkiangensis in the world. It will provide a basis for research into the occurrence, distribution of V. nivea on Korla Pear.

First report of Diplodia mutila associated with Botryosphaeria canker and dieback of apple trees in Xinjiang, China.

Apple (Malus pumila Mill.) is an important fruit crop in Xinjiang, China. In September 2021, apple tree canker was observed in a 21-year-old commercial apple orchard cv. Fuji in Xinjiang (38°17'51.43"N, 77°9'50.81"E) , northwest of China. Of the 200 plants surveyed, 25% were symptomatic. The diseased trees showed branch dieback and cankers. The cankers observed on the wood were sunken, shriveled, and discolored. After the bark was peeled off, the diseased wood was dark brown, and the necrosis was obvious on the cross-section of the diseased branch. To identify the causal agent, five symptomatic trees were collected and analyzed in the laboratory. Apple wood samples (0.5×0.5 cm) were surface-disinfected with 1% v/v sodium hypochlorite and 75% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA), and incubated in the dark at 25 °C for 5 days. Conidia were induced on sterilized pine needles covered with 2% w/v water agar under near-UV light. The colonies of five isolates were white to gray with sparse aerial mycelium that gradually became dark olive green in the later stage. Conidia were initially hyaline but becoming brown at maturity, 1-septate, oval, rounded at both ends, and with dimensions of 24.9-32.1 × 15.1-21.5 µm (n =50) and the aspect ratio of 1.6. Based on the cultural and morphological features of Phillips (2002), the isolates were identified initially as Diplodia mutila (Fr. : Fr.) Mont. To confirm species identification, genomic DNA was extracted from the representative isolate SC-8A. The primer ITS1/ITS4, EF1-728F/EF1-986R and BT2a/BT2b were used to amplify the rDNA sequences of, respectively, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1-α) gene, and a portion of beta-tubulin (tub2) gene. The nucleotide sequences indicated ≥99% identity to D. mutila (CBS 112553) for three DNA regions. Consensus sequences were deposited in GenBank. as accession numbers OM618108, OM676657 and OM676658 for ITS, EF1-α and tub2, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate SC-8A on one year old branches of cv. Fuji (n=5). Wounds were created in the middle of the branches using a sterilized hole punch (5mm diameter) and were immediately inoculated with mycelial plugs of the same diameter. For the control treatment, sterile agar plugs were used (n=5) in the branches. The inoculated and control branches were wrapped with sterile parafilm. On the 10th day after inoculation, canker lesions appeared on the inoculated branches, but no lesions were observed in the negative control. D. mutila was re-isolated from 100% of the inoculated shoots and was not re-isolated from any of the negative controls, the Koch's postulates were met. Previously, D. mutila has been reported in Canada (Úrbez-Torres et al., 2016), Argentina (Lódolo et al., 2022) and Chile (Díaz et al., 2022) causing Botryosphaeria canker and dieback in apples. To our knowledge, this is the first report of D. mutila causing Botryosphaeria canker and dieback in apple trees in China.

Genome Sequence of the Agrobacterium salinitolerans DG3-1 Isolated from Cotton Roots.

Agrobacterium salinitolerans DG3-1 is an endophytic bacterium isolated from cotton root tissue. Our previous work has shown that it can inhibit the growth of Fusarium and Verticillium wilt pathogens as well as increase the chlorophyll content of cotton leaves. Here, we reported the complete genome sequence of strain DG3-1, which was analyzed by sequence reads generated from Nanopore PromethION and Illumina NovaSeq PE150 platforms. This genome sequence could be used to clarify the possible mechanism of DG3-1 at the gene level.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.