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.

Root-Associated Microbiota Response to Ecological Factors: Role of Soil Acidity in Enhancing Citrus Tolerance to Huanglongbing.

The citrus orchards in southern China are widely threatened by low soil pH and Huanglongbing (HLB) prevalence. Notably, the lime application has been used to optimize soil pH, which is propitious to maintain root health and enhance HLB tolerance of citrus; however, little is known about the interactive effects of soil acidity on the soil properties and root-associated (rhizoplane and endosphere) microbial community of HLB-infected citrus orchard. In this study, the differences in microbial community structures and functions between the acidified and amended soils in the Gannan citrus orchard were investigated, which may represent the response of the host-associated microbiome in diseased roots and rhizoplane to dynamic soil acidity. Our findings demonstrated that the severity of soil acidification and aluminum toxicity was mitigated after soil improvement, accompanied by the increase in root activity and the decrease of HLB pathogen concentration in citrus roots. Additionally, the Illumina sequencing-based community analysis showed that the application of soil amendment enriched functional categories involved in host-microbe interactions and nitrogen and sulfur metabolisms in the HLB-infected citrus rhizoplane; and it also strongly altered root endophytic microbial community diversity and structure, which represented by the enrichment of beneficial microorganisms in diseased roots. These changes in rhizoplane-enriched functional properties and microbial composition may subsequently benefit the plant's health and tolerance to HLB disease. Overall, this study advances our understanding of the important role of root-associated microbiota changes and ecological factors, such as soil acidity, in delaying and alleviating HLB disease.

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.

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.

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.