Characterization of Bacillus amyloliquefaciens BA-4 and its biocontrol potential against Fusarium-related apple replant disease.

Apple replant disease (ARD), caused by Fusarium pathogens, is a formidable threat to the renewal of apple varieties in China, necessitating the development of effective and sustainable control strategies. In this study, the bacterial strain BA-4 was isolated from the rhizosphere soil of healthy apple trees in a replanted orchard, demonstrating a broad-spectrum antifungal activity against five crucial apple fungal pathogens. Based on its morphology, physiological and biochemical traits, utilization of carbon sources, and Gram stain, strain BA-4 was tentatively identified as Bacillus amyloliquefaciens. Phylogenetic analysis using 16S rDNA and gyrB genes conclusively identified BA-4 as B. amyloliquefaciens. In-depth investigations into B. amyloliquefaciens BA-4 revealed that the strain possesses the capacity to could secrete cell wall degrading enzymes (protease and cellulase), produce molecules analogous to indole-3-acetic acid (IAA) and siderophores, and solubilize phosphorus and potassium. The diverse attributes observed in B. amyloliquefaciens BA-4 underscore its potential as a versatile microorganism with multifaceted benefits for both plant well-being and soil fertility. The extracellular metabolites produced by BA-4 displayed a robust inhibitory effect on Fusarium hyphal growth and spore germination, inducing irregular swelling, atrophy, and abnormal branching of fungal hyphae. In greenhouse experiments, BA-4 markedly reduced the disease index of Fusarium-related ARD, exhibiting protective and therapeutic efficiencies exceeding 80% and 50%, respectively. Moreover, BA-4 demonstrated plant-promoting abilities on both bean and Malus robusta Rehd. (MR) seedlings, leading to increased plant height and primary root length. Field experiments further validated the biocontrol effectiveness of BA-4, demonstrating its ability to mitigate ARD symptoms in MR seedlings with a notable 33.34% reduction in mortality rate and improved biomass. Additionally, BA-4 demonstrates robust and stable colonization capabilities in apple rhizosphere soil, particularly within the 10-20 cm soil layer, which indicates that it has long-term effectiveness potential in field conditions. Overall, B. amyloliquefaciens BA-4 emerges as a promising biocontrol agent with broad-spectrum antagonistic capabilities, positive effects on plant growth, and strong colonization abilities for the sustainable management of ARD in apple cultivation.

Barriers associated with the public use of sports facilities in China: a qualitative study.

BACKGROUND: Sports and recreational facilities provide an important community resource where physical activity can be promoted among local residents. However, in China, many sports facilities are not fully open to the public and are often underutilized as essential public services. The purpose of this study was to examine the barriers to public entry from the facility management point of view. METHODS: A qualitative study informed by constructivist grounded theory. Individual interviews were conducted with executive managers, marketing staff, and facilities management personnel (n = 15). We took an inductive approach to data synthesis utilizing procedural three-level coding, and used the electronic data management program (NVivo12) to organize the data analysis process. RESULTS: The 15 participants discussed factors that impacted the sports facilities' capacity to serve the public. Four key themes emerged from data synthesis: (a) Policy-related restrictions, (b) Management-related factors, (c) Service-related factors, and (d) Supervision factors. Specific barriers affecting facilities' availability and accessibility involved policies and regulations, administrative pressure, institutional mechanisms, ideas, a professional team, practical ways, content development, schedule setting, spirit building, assessment and supervision, crisis management, and public evaluation. CONCLUSION: Findings from this qualitative study provided theoretical ground and empirical support for future research aimed at making sports facilities more accessible to the public in order to maximize physical activity and meet the objectives of the national fitness blueprint in China.

Malus asiatica as a natural host of apple scar skin viroid in China.

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, fragments < 18 nt and > 26 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.

Coinfection of Two Mycoviruses Confers Hypovirulence and Reduces the Production of Mycotoxin Alternariol in Alternaria alternata f. sp. mali.

Alternaria leaf blotch caused by Alternaria alternata apple pathotype (Alternaria mali) is an important fungal disease that affects the production of apples worldwide. Mycoviruses harbored in plant pathogenic fungi can confer hypovirulence in their hosts and have attracted widespread attention as potential biocontrol tools. In this study, the coinfection of two mycoviruses, named A. alternata chrysovirus 1 strain QY2 (AaCV1-QY2) and A. alternata magoulivirus 1 (AaMV1), respectively, were isolated from A. alternata f. sp. mali strain QY21. Sequence analyses revealed that AaCV1-QY2 virus belonged to the genus Betachrysovirus and AaMV1 virus belonged to the genus Magoulvirus. These two mycoviruses were found to be associated with hypovirulence in A. alternata, among which AaCV1-QY2 might play a relatively leading role. Because the elimination of AaMV1 from the strain QY21 does not affect the hypovirulence trait, which indicates that the virus AaCV1-QY2 can independently induce slow growth and reduce host virulence. Moreover, the presence of viruses decreased the accumulation of the mycotoxin alternariol (AOH) in A. alternata strains. Intriguingly, AaCV1-QY2/AaMV1 mycoviruses can be horizontally transmitted to other A. alternata strains, and this coinfection can promote the interspecific transmission efficiency of AaCV1-QY2. To our knowledge, this study reports the first description of the member of Chrysovirus is related to hypovirulence in Alternaria spp. that facilitates the development of biocontrol measures of A. mali Roberts.

First report of apple root rot caused by Phytopythium vexans in China.

Apple production is of great economic importance in the fruit industry of China, where Yunnan Province is considered as a major producing area. A survey was conducted to identify apple trees that were problematic from March to November 2020 in Yunnan Province. Symptoms included smaller yellowing leaves, fewer sprouts per branch, browning and necrosis of the roots and lower parts of the stem bark, and wilting. 20% to 45% of apple trees were found infected and randomly scattered in the surveyed orchards. A total of 110 soil samples were collected from the root area of symptomatic apple trees in Tuanjie Town of Kunming City, Zhaotong City, and Malong District of Qujing City in Yunnan Province. Two grams of each soil sample was suspended in 400 ml of sterile water for three days and each soil extract was baited with two apple leaves (Red Fuji's). Following the baiting, those leaves were cut into 10 pieces (5mm×5mm), surface-sterilized with 70% ethanol for 30 seconds, rinsed three times with sterile water, and then air-dried. Each leaf piece was placed in a Petri dish with the oatmeal agar medium containing PCNB 20 mg/ml, rifampicin 20 mg/ml, and then incubated at 25℃ in the dark for 3 days. A mycelial agar plug was picked from the edge of the colonies and transferred to a fresh Potato Dexrose Agar (PDA) plate. Seventy colonies with similar growing characteristics were isolated from the 110 soil samples. Three isolates were retained for further analysis and named XLD8-1, SD1, and YF2. After being cultivated on PDA plates and incubated at 25℃ in the dark for 4 days, their colonies were rose petal-type and white with dense aerial hyphae (Fig 1, A). In ten days of incubation, oogonium measuring 24.55 ± 1.9µm × 20.27 ± 2.3µm and sporangia measuring 21.65 ± 1.3µm × 19.35 ± 1µm were observed (Fig 1, C, D). The total DNA of the isolates was extracted and amplified using three pairs of primers, ITS1/ITS4 (White et al. 1990), LROR/LR7 (LSU) (Vilgalys R, et al. 1990), and FM58/FM66 (COXⅡ) (Martin F N. 2000). The sequences were uploaded to GenBank (Accession No. OL960234, OK037658, OK052604 for ITS, OL960388, OM838413, OM838314 for LSU, and OM962847, OM962848, OM962849 for COXⅡ). ITS sequences of the three isolates (XLD8-1, SD1, YF2) showed 99.87%,99.87%, 99.87% similar to Pp. vexans (Accession No. AB468784, AB468784, and AM701801). LSU sequences of the three isolates showed 99.92%, 99.72%, 100% similar to Pp. vexans (Accession No. EF426541, MT729990, and EF426541). COXⅡ sequences of the three isolates showed 100%, 99.81%, 99.81% similar to Pp. vexans (Accession No. GU133560). Based on the sequence similarity and morphology, the isolates were identified as Phytopythium vexans. Koch's postulates were conducted by wounding the bases of 3 apple seedlings (1-year-old Red Fuji's) with a cork borer. A plug of mycelium of the isolate XLD8-1 grown on PDA plates was placed on each wound (Fig 1, B). Controls were set up to use sterile agar plugs as an inoculum. Seedlings have incubated an incubator at 23-26°C under the alternating light and dark intervals, 12-hours of each. In 15 days, after were inoculated with XLD8-1 the roots and lower part of the stem bark of those seedlings became brownish and necrotic, and their epidermis was easily sloughed off (Fig 1, E-G). The pathogen isolated from the necrotic root tissues were identical to the isolate XLD8-1. Symptoms of apple growth decline caused by Pp. vexans were reported in Morocco (Jabiri Salma, et al. 2021). This experiment verified that Pp. vexans causes root rot of apple. In China, Fusarium sp. is usually considered the main pathogen causing apple root rot. However, the discovery of large numbers of apple trees that were infected by Pp. vexans in Yunnan Province and the confirmation of pathogenicity of Pp. vexans on apple seedlings have demonstrated for the first time that Pp. vexans could cause apple root rot as Fusarium spp does and become an incoming threat to the apple industry, which lays the foundation for study on the disease epidemiology and integrated management of apple root rot in China. References: Jabiri Salma, et al. 2021. Microorganisms, doi:10.3390/MICROORGANISMS9091916. Martin, F. N. 2000. Mycologia, 92(4), 711-727. Vilgalys R., et al. 1990. Journal of Bacteriology, 172:4238-4246 White, T. J., et al. 1990. PCR Protocols: a guide to methods and applications, 18: 315.

Guttation Fluids Containing Pseudomonas amygdali pv. lachrymans Are a Potential Source of Secondary Infection in Cucumber.

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.

Raw data of laboratory toxicity and field control effect of different fungicides on Colletotrichum fructicola.

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).

The Influence of Lower Temperature Induction of Valsa mali on the Infection of Apple Trees.

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.

Monitoring of Fusarium Species and Trichothecene Genotypes Associated with Fusarium Head Blight on Wheat in Hebei Province, China.

To clarify the changes in field populations of Fusarium head blight (FHB) pathogens over a decade, Fusarium species and trichothecene genotypes associated with FHB on wheat were monitored in Hebei province during the periods 2005-2006 and 2013-2016. Fusarium species determination was carried out by morphological identification, species-specific amplification and partial translation elongation factor (TEF-1α) gene sequencing. Trichothecene genotype prediction was carried out by primers 3CON/3NA/3D15A/3D3 or Tri13F/Tri13R, Tri303F/Tri303R and Tri315F/Tri315R. A total of 778 purified Fusarium isolates were recovered from 42 sampling sites in 17 counties during the period 2005-2006 and 1002 Fusarium isolates were recovered from 122 sampling sites in 65 counties during the period 2013-2016. F. graminearum was the predominant pathogen recovered during the periods 2005-2006 and 2013-2016. However, the pathogen composition differed slightly between the two periods. In 2005-2006, 752 out of 778 (96.7%) of the isolates belonged to F. graminearum. Two were identified as F. culmorum. Five other Fusarium species were also recovered, F. equiseti, F. verticillioides, F. proliferatum, F. subglutinans and F. chlamydosporum, with lower recoveries of 0.4%, 0.8%, 0.8%, 0.1% and 1.0%, respectively. Trichothecene genotype prediction showed that all the 752 F. graminearum isolates were of the 15-ADON genotype. Five Fusarium species were recovered from samples collected over the period 2013-2016. F. graminearum was again the predominant pathogen with an isolation frequency of 97.6%. F. pseudograminearum, F. asiaticum, F. culmorum and F. negundis were also isolated at a recovery of 1.4%, 0.7%, 0.2% and 0.1%, respectively. For the 2013-2016 isolates, 971 of the 978 F. graminearum strains were 15-ADON whereas seven isolates were of the 3-ADON type. All seven F. asiaticum isolates were of the NIV type and fourteen F. pseudograminearum isolates were classified as 3-ADON. F. pseudograminearum was first isolated from FHB in Hebei in 2013. Although the recovery of F. pseudograminearum is still low, it represents a small shift in the pathogen composition and trichothecene genotypes associated with FHB in Hebei province. As Fusarium crown rot of wheat caused by F. pseudograminearum is an increasing problem in Hebei province, it is appropriate to monitor the role of F. pseudograminearum in FHB in the future.

Latent Infection of Valsa mali in the Seeds, Seedlings and Twigs of Crabapple and Apple Trees is a Potential Inoculum Source of Valsa Canker.

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.

[Identification of Host Factors Interacting with the Movement Protein of Apple Chlorotic Leaf Spot Virus by Yeast Two-Hybrid System].

In order to identify host factors which interact with the movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV), ACLSV MP was cloned into the bait vector pGBKT7 and used to screen a cDNA library of Malus sylvestris cv. R12740-7A, which had previously been constructed by yeast two-hybrid sequencing transformation. The protein functions of the identified host factors were determined according to their gene annotations in GenBank. The result showed that the bait plasmid pGBKT7-MP showed no virulence or self-activating effect on yeast strain Y2H Gold. Sixty-nine interactor proteins were identified, which were divided into the following 10 classes according to their described functions: hydrolases; pathogenesis-related proteins; DNA binding proteins; phosphatases; ligases; proteins with catalytic activity; phenylalanine ammonialyases; peroxidases; NAD binding proteins; and proteins of unknown function. Bioinformatic analysis of gene homology suggested that phosphatases, pathogenesis-related proteins and glyceraldehyde-3-phosphate dehydrogenase A may play an important role in the interaction between virus and host. This study may provide a theoretical basis for the further study of viral pathogenesis and virus-host interaction mechanisms.