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Guttation is a common feature of cucumber leaves under high relative humidity conditions; however, little is known about the role of guttation in the transmission of Pseudomonas amygdali pv. lachrymans, which is the pathogen of cucumber angular leaf spot disease. In this study, experimental evidence for the transmission of P. amygdali pv. lachrymans inside cucumber plants and through guttation was provided, and the results proved that P. amygdali pv. lachrymans can be transmitted from the bottom leaf to the upper leaves inside the plant and excreted from the upper leaves through guttation. After that, the third leaf of cucumber was inoculated with P. amygdali pv. lachrymans bacterial suspension, P. amygdali pv. lachrymans was detected on the fifth leaf, the petiole, and the stem and in guttation drops. Healthy cucumber seedlings were infected by P. amygdali pv. lachrymans in the guttation droplets, indicating that guttation fluids containing P. amygdali pv. lachrymans could become a potential source of secondary infection. The results from this study verified the hypothesis that guttation is a potential route for P. amygdali pv. lachrymans excretion from cucumber plants and may be a source of secondary transmission under high relative humidity.

Apple valsa canker (AVC), caused by Valsa mali, is one of the most important diseases of apple trees in China. AVC occurred severely along with cold winter or cold spring. However, the effect of lower temperature on V. mali is poorly understood. This study evaluated the influence of lower temperature pretreatment of V. mali on the infection of apple twigs and leaves. The results showed that exposing V. mali to lower temperatures (between –10°C and 10°C) for more than 18 h significantly increased the disease severity of apple leaves and twigs, with a higher lesion area ratio (LAR), lesion length, and disease incidence (DI) than that at 25°C. In addition, cold treatment ranging from –5°C to 10°C promoted colony growth. Meanwhile, the relative expression of four cell wall degrading enzyme (CWDE)-related genes pretreated at –5°C and 5°C were significantly higher than that at 25°C. The results indicated that the virulence of V. mali mycelium is sensitive to lower temperatures. After sensing lower temperature changes, V. mali can adjust its infection of apple trees by regulating the expression of pathogenicity gene and growth rate. Spring has very frequent temperature changes, and V. mali is highly invasive in this season. Therefore, more attention should be paid in spring to protecting apple trees from infection of V. mali, by reducing pruning wound formation in spring and applying protective agents to pruning wounds in time.

There are few studies of cucumber roots at the seedling stage infected by Pseudomonas amygdali pv. lachrymans (Pal). We used growth chamber assays to determine the infection of cucumber seedling roots by Pal following artificial substrate inoculation. Symptoms on cucumber roots, leaves, and stems were observed over time. We used real-time fluorescent quantitative PCR (qPCR) to obtain accurate numbers of Pal copies in infected roots, and then statistically assessed the dynamics of Pal copies in roots, stems, and leaves. Results indicated that Pal not only caused root decay, but also damaged the whole plant. Following root infection, the bacteria were transmitted through stems to the leaves. Cucumber seedlings in a moist planting substrate showed lodging, leaf chlorosis and wilt, and rotted roots. This study provides evidence that in moist substrate, Pal can infect cucumber roots and be transmitted throughout the host plant. In addition, the results also provide a foundation for further research on the transmission and proliferation of Pal in cucumber plants under field conditions.

Malus asiatica (Rosaceae, Malus) is a small deciduous tree, which has been cultivated in China more than 450 years (Jin, 2019). M. asiatica is deeply favored by consumers because of its sweet taste and high nutritional attributes, rich in vitamins, minerals and dietary fiber (Xue et al, 2013). Although the M. asiatica annual output is nearly 30 000 kg, it still cannot meet the market demand in China (Jin, 2019). In August 2021, the virus-like symptom such as colored spots on fruit epidermis of M. asiatica were observed in an orchard of Langfang (38°42'16.88″N, 116°39'15.23″E) of Hebei province, China. To investigate whether this symptom is related to virus infection, the symptomatic sample was subjected to small RNA sequencing. Total RNA was extracted from branch bark of a symptomatic tree using an RNAprep Pure Plant Kit (TianGen, China), The extracted RNA was used to construct a small RNA library using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1), (NEB, USA), then the resulting library was sequenced using Illumina novoseq 6000 (Illumina, USA) at Tianjin Novogene company (China). A total of 14,685,616 sequence reads were obtained. After filtering the low-quality reads, polyA, adaptor contaminants, fragments18 nt and26 nt, and reads matching apple genome, the number of reads reduced to 392,883. Finally, assembly of these clean reads generated 225 non-redundant contigs with Velvet software and 55 assembled contigs were aligned to Refseq viral database of NCBI by Bowtie software. One viral contig with length of 329 nt showed 98.48% significant similarity to genome sequences of Hohhot isolate of ASSVd (ASSVd-Hohhot) (GenBank Accession No. MZ476527.1) (Yuan et al, 2022). We then used a specific primer pair (ASSVd-F: 5'-G G T A A A C A C C G T G C G G T T C C-3'; ASSVd-R: 5'-G G G A A A C A C C A A T T G T G T T T T A-3') for reverse transcription (RT)-PCR to amplify the genome sequence of ASSVd. A 330 bp amplified product was cloned into the pGEM-T easy vector (Promega, USA), then sequenced by Sanger sequencing using T7 primer by Sangon Biotech (Shanghai) Co., Ltd. in China. The sequence of ASSVd has been deposited in the GenBank datebase (GenBank Accession No. ON093255). Blast analysis showed that the sequence had highest identity (326/330, 98.79%) with ASSVd-Hohhot (GenBank Accession No. MZ476527.1) (Yuan et al, 2022). To confirm the pathogenicity of ASSVd, fifteen healthy cucumber seedlings were inoculated mechanically with the extracts of ASSVd-infected branch bark of M. asiatica. There were no obvious symptoms were observed at 14 days post inoculation (dpi), however, the result of RT-PCR and Sanger sequencing showed four cucumber samples were positive for ASSVd. In addition, another 19 randomly collected M. asiatica samples with or without clear symptoms from Langfang were detected by RT-PCR, and ten (52.6%) of them were confirmed the presence of ASSVd. And all ten positive samples were symptomatic, while nine nonsymptomatic M. asiatica samples tested negative. The positive amplicons were cloned into the pGEM-T easy vector and sequenced using T7 primer by Sanger sequencing. All of the sequences were essentially identical to one another (GenBank Accession No. ON093255), which indicates that the positive samples are indeed ASSVd infected. To the best of our knowledge, this is the first report of ASSVd infection in M. asiatica, which expands our understanding of the host range of ASSVd.

Apple replant disease (ARD) occurs frequently in old orchards and is an important disease that limits apple production in China. Fusarium oxysporum is one of the causal agents of ARD and its pathog...

Laboratory toxicity test and field control effect test are important experiments to evaluate the effect of pesticides on disease control. The safety of pesticide application depends on whether the pesticide residue on the fruit complies with the limits related to the good agricultural practices which were assessed by the legislative framework. In addition, many factors may affect the control effect of fungicides in the field experiment, among which one of the most important factors is the precipitation during the application process in the growing season. In this study, raw data of the laboratory toxicity of different fungicides to Colletotrichum fructicola, disease severity after treatment with different agents, the pesticide residue on the fruit after preharvest application, and the precipitation in the growing season are provided. In addition, this study also introduced the method and calculation process of raw data processing. We hope that these raw data will serve as a reference for other researchers who are studying the prevention and control of Glomerella leaf spot (GLS).

Guttation is a common feature of cucumber leaves under high relative humidity conditions; however, little is known about the role of guttation in the transmission of

A real-time quantitative PCR assay using a species-specific primer pair was developed to rapidly and accurately quantify Valsa mali, the causative pathogen of apple Valsa canker (AVC), in crabapple seeds, crabapple seedlings, apple twigs and apple seeds. Surveys were conducted in different regions, and crabapple or apple seeds were collected for V. mali detection by qPCR assay. Our results showed that 12.87% to 49.01% of crabapple seeds collected from different regions were positive for V. mali. The exopleura and endopleura were the two major areas of V. mali infection in crabapple seeds. The presence of V. mali infection in crabapple seeds was also confirmed by a high-throughput sequencing approach. With the growth of crabapple seedlings, the concentration of V. mali gDNA in crabapple seedlings gradually increased until eight or more leaf blades emerged. One-year-old twigs from an apple scion nursery were infected with V. mali, and only apple seeds from infected apple trees showing evident Valsa canker symptoms carried V. mali. In conclusion, this study reports that crabapple seeds and apple seeds carried V. mali as latent inoculum sources. V. mali infected not only apple tissues but also crabapple seedlings, which are the rootstocks of apple trees. This study indicated that the inoculum sources for AVC vary. Application of a novel qPCR assay can potentially improve the accuracy of early diagnosis, and is helpful to reveal the epidemic regularity of AVC.

Apple Glomerella leaf spot (GLS) is caused by Colletotrichum fructicola, which causes severe leaf spot symptoms and leads to early defoliation, resulting in considerable economic losses. Since few efficient chemical control strategies were yet available in China, the aim of this work was to establish the basis of a control strategy for GLS. In the present study, we determined in vitro toxicity of three commercial fungicides prochloraz, pyraclostrobin and tebuconazole against mycelial growth and conidia germination of C. fructicola. The EC50 values of the three fungicides on mycelial growth ranged from 0.02 to 0.55 μg ai mL−1, and that on conidia germination ranged from 2.01 to 3.02 μg ai mL−1. Two consecutive years of field experiment was conducted in 2017 and 2018 in four apple orchards of Shandong province in China for further verifying the control effect of these three agents. The four apple orchards that planted with susceptible cultivar ‘Gala’ use two training systems, one is the slender pyramid training system (SP), and the other is the slender spindle training system (SS). Besides the target agent treatment, Bordeaux mixture was also sprayed for one or two times in a growing season. Synthetic fungicide pyraclostrobin sprayed at the dose 810.00, 405.00, and 202.50 g ai ha−1 in 2017, and 810.00, 405.00, and 303.75 g ai ha−1 in 2018. Tebuconazole was applied at the dose 580.50 g ai ha−1 in 2017, and prochloraz was applied at the dose 911.25 g ai ha−1 in 2018. Results showed that the treatment of pyraclostrobin at 810.00 and 405.00 g ai ha−1 combined with Bordeaux mixture have good control effect. The application frequency of fungicides in SS training system orchard is one to two times less than that in SP training system orchard, but the control effect of chemical agents in the former is greater than or equal to the latter. The final residues of all pesticides were below the Chinese national standards for pesticide residues. In conclusion, the application strategy of spraying pyraclostrobin combined with Bordeaux mixture can effectively prevent and control GLS caused by C. fructicola, and reasonable preharvest application of the fungicide will not cause excessive pesticide residues.

Apple canker caused by Valsa mali is a destructive disease that causes serious economic losses in China. In this study, the dynamics of release of conidial masses (cirri) in apple orchards were studied in different seasons, and in laboratory tests the effects of temperature on conidial germination were determined. Using artificial inoculations in the field, the influence of the pruning process on year-round disease development, and the possibility of the pruning saws carrying conidia of V. mali were tested to determine disease transmission in the winter months. Conidial masses released from pycnidia demonstrated strong adaptability to the changing environment. Conidial exudation from pycnidia could last up to a year and take place even in the winter, in the period when the bark was wet. The germination rate of conidia reached 90 % after 6 days incubation at 5 °C and 50 % after 19 days at 0 °C. Infection rates of conidia in different seasons were determined in a 3-year experiment. Fresh pruning wounds were more susceptible to infection than old wounds, and conidia were able to infect apple trees and induce canker lesions at different times of the year, and especially in winter. Pruning in early winter may promote heavy infections of apple by V. mali and could play an important role for the spread of the disease.

Blueberry (Vaccinium corymbosum) has developed rapidly in China over the past 20 years because of its perceived health and nutritional benefits. Blueberry stem blight, caused by fungi in the family Botryosphaeriaceae, is the most destructive disease affecting blueberry production and quality worldwide. A field survey of 20 blueberry plantations in eight provinces across China was conducted to determine the occurrence and distribution of Botryosphaeriaceae species. In total, 69 Botryosphaeriaceae isolates associated with blueberry twig dieback and stem blight were identified based on morphological characteristics and analyses of nucleotide sequences of three genomic regions: the internal transcribed spacer region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). Three species were identified, including: Botryosphaeria dothidea, Neofusicoccum parvum and Lasiodiplodia theobromae. Botryosphaeria dothidea and N. parvum were the most prevalent species and were widely distributed in all blueberry-growing regions. In contrast, L. theobromae was only found in southeastern regions. Koch’s postulates showed all three species to be pathogenic when inoculated with mycelial plugs on detached and intact blueberry stems (cv. Bluecrop). Pathogenicity tests showed N. parvum and L. theobromae to be more virulent than B. dothidea. To our knowledge, this study is the first comprehensive description for morphology, phylogenetic analysis, and distribution of Botryosphaeriaceae spp. as causal agents of blueberry stem blight in China.

Apple chlorotic leaf spot virus (ACLSV) is one of the most economically important latent viruses infecting apple in China. This is the first report of the almost complete nucleotide sequence and the characterization of the genome of a Chinese isolate (ACLSV-MS, GenBank Accession Number KC847061) from apple. Based on the genome nucleotide sequence, ACLSV-MS showed the highest identity (99.4%) to isolate ACLSV-B6 (GenBank Accession Number AB326224) from apple in Japan and the least identity (69.5%) with isolate TaTao5 (GenBank Accession Number: EU223295) from peach in the USA. The occurrence and distribution of ACLSV in China were also recorded. Three hundred and twenty-seven apple samples (40 different cultivars) collected from 56 sites in 13 provinces of China were tested by RT-PCR. The virus was detected in all regions surveyed (the provinces of Heilongjiang, Liaoning, Hebei, Beijing, Henan, Shanxi, Shaanxi, Shandong, Gansu, Ningxia, Xinjiang, Sichuan and Yunnan), with an average incidence of 69.7%. The positive samples in Heilongjiang province were highest with an incidence of 100% followed by Henan province with an incidence of 86.7%. The positive samples in Liaoning and Shanxi were the lowest with an incidence of 50%. The occurrence of virus in five common cultivars was determined. The percentage of ACLSV was highest in cv. Gala with an incidence of 33.3%, while lowest in cv. Starking with an incidence of 18.2%. It was also found in younger (≤20 years) apple orchards the occurrence of ACLSV decreased with the increase of tree age, but when trees were more than 20 years old, the occurrence of ACLSV increased. This is the first extensive survey in the last decade in China for monitoring ACLSV, which provides important information for ACLSV control in China.

The fungal pathogen Botrytis cinerea Pers. causes severe rotting on tomato fruits during storage and shelf life. As a biological control agent, endophytic bacterium was regarded as an effective alternative to chemical control. Out of 238 endophytic bacterial isolates, three strains (EB-15, EB-28, and EB-122) isolated from Lycopersicum esculentum Mill., Speranskia tuberculata (Bge.) Baill, and Dictamnus dasycarpus Turcz. respectively were found to be strongly antagonistic to the pathogen in vitro and were selected for further in vivo tests. One endophytic bacterium strain, encoded EB-28, was selected from the three in vivo tested isolates. The inhibitive rate of EB-28 reached 71.1% in vitro and 52.4% in vivo. EB-28 was identified as Bacillus subtilis according to its morphological, physiological, and biochemical characteristics and 16S rDNA sequence analysis.

Fusarium graminearum is the main causative agent of cereal scab and maize cob rot in China. Two hundred and fifty-five F. graminearum isolates were obtained from wheat, barley and maize from Hebei, Heilongjiang and Hubei, provinces with distinct climate conditions and cropping systems. The isolates were confirmed to be F. graminearum by polymerase chain reaction (PCR) assay using F. graminearum species-specific primers Fg16F/Fg16R. Two populations, 7C1 and 6A5, were identified to exist in China. The 6A5 population was predominant in Hubei in central China along the Yangtze River, whereas the 7C1 population was predominant in Heilongjiang and Hebei in northern China. Based on sequences of Tri13 and Tri3, genes involved in the mycotoxin biosynthetic pathway, PCR assays were used to detect 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV) chemotypes. All three chemotypes of F. graminearum were identified, with 15-ADON chemotype predominating overall. A greater proportion of 3-ADON chemotype was found in Hubei, whereas a greater proportion of 15-ADON was found in Heilongjiang and Hebei.

Six extracts from plant material (Galla chinensis, Potentilla erecta, Rheum rhabarbarum, Salviae officinalis, Sophora flavescens, and Terminalia chebula) were tested for controlling effects against the infection of Phytophthora infestans on detached potato leaves, seedlings, and tuber slices. On detached leaves, G. chinensis (2%), R. rhabarbarum (rhizome, 2%) and S. flavescens (2%) extracts showed a significant control effect, with a control efficacy of 96.67%, G. chinensis was the best. On seedlings R. rhabarbarum (rhizome, 2%) showed the best inhibiting effect, followed by S. flavescens (2%), T. chebula (1%), and G. chinensis (2%). The control efficacies were 91.67%, 75.00%, 70.24%, and 64.29%, respectively on the seventh day after inoculation. However, on potato slices, none of the plant extracts showed effective protection against infection and sporangia production by P. infestans. The reason was analyzed and the potential for developing a natural fungicide based on these plant materials was discussed.