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.

Purine metabolism in plant pathogenic fungi.

In eukaryotic cells, purine metabolism is the way to the production of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and plays key roles in various biological processes. Purine metabolism mainly consists of de novo, salvage, and catabolic pathways, and some components of these pathways have been characterized in some plant pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae and wheat head blight fungus Fusarium graminearum. The enzymatic steps of the de novo pathway are well-conserved in plant pathogenic fungi and play crucial roles in fungal growth and development. Blocking this pathway inhibits the formation of penetration structures and invasive growth, making it essential for plant infection by pathogenic fungi. The salvage pathway is likely indispensable but requires exogenous purines, implying that purine transporters are functional in these fungi. The catabolic pathway balances purine nucleotides and may have a conserved stage-specific role in pathogenic fungi. The significant difference of the catabolic pathway in planta and in vitro lead us to further explore and identify the key genes specifically regulating pathogenicity in purine metabolic pathway. In this review, we summarized recent advances in the studies of purine metabolism, focusing on the regulation of pathogenesis and growth in plant pathogenic fungi.

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.

Ultrasensitive detection of PCB77 based on Exonuclease III-powered DNA walking machine.

In view of the urgent need to determine polychlorinated biphenyls (PCBs) in the environment, we report a simple and sensitive electrochemical aptasensor to detect 3,3',4,4'-tetrachlorobiphenyl (PCB77) based on Exonuclease III-powered Deoxyribonucleic Acid (DNA) walking machine using poly (diallyldimethylammonium chloride) (PDDA), which was functionalized hollow porous graphitic carbon nitride/ Ni-Co hollow nanoboxes/ Au-Pd-Pt nanoflowers composite material. Upon the addition of PCB77, the specific binding between PCB77 and the aptamer (Apt) could trigger the Exo III-assisted cyclic amplification process and release unlocking probes to deblock the Swing arm/Blocker duplex. Finally, the hybridized hairpin 3 (HP3), a short oligonucleotide, was left on the electrode via Exo III digestion of hybridized HP2, and thus a strong methylene blue (MB) signal was obtained. As expected, the proposed aptasensor exhibits exceptional PCB77 detection performances with a very low detection limit of 5.13 pg/L and a wide linear range of 0.01-100 ng/L based on the calibration curve. Moreover, the aptasensor presents a high level of selectivity and stability, with an acceptable degree of reproducibility. The results of this study have indicated that the proposed aptasensor has great potential application prospects, as demonstrated by its successful use in real environmental water samples.

Trichothecium roseum Enters 'Fuji' Apple Cores Through Stylar Fissures.

Apple core rot, an economically important disease worldwide, appears both before and during harvest. Current gaps in understanding of the infection cycle impede progress toward more effective management of this disease. The fungus Trichothecium roseum is the main pathogen of core rot on apple in China. In this study, we used fluorescent labeling to trace colonization of T. roseum in floral tissues, characterizing routes of penetration to the core of 'Fuji' apples. T. roseum infected petals, anthers, filaments, stigmas and separated styles of flowers, and floral debris served as inoculum for core infection. In field inoculations, T. roseum entered styles initially through stylar fissures and colonized pluricellular hairs of these fissures during early stages of fruit development. Subsequently, hyphae grew along the extending fissures, which are continuations of stylar fissures located between stylar bases and carpel cavities. The hyphae remained in the extending fissures from mid-June to late July. When fruit developed an open sinus in late July, the sinus eventually fused with extending fissures and carpel cavities in late August, hyphae invaded carpel cavities, and ultimately fruit flesh via cracks on carpel cavity walls. Our results revealed for the first time the routes by which T. roseum penetrates apple fruit, and provided significant insights for strategic management of core rot.

High Humidity and Age-Dependent Fruit Susceptibility Promote Development of Trichothecium Black Spot on Apple.

Fruit bagging is a widely used orchard practice in China. Trichothecium black spot (TBS) is a disease highly associated with the fruit bagging. In this study, we characterized in vitro factors affecting the causal agent, Trichothecium roseum, and TBS development and infection histology on field-bagged apple fruit in situ. Under in vitro conditions, conidial germination required exogenous nutrients, and the germination rate was significantly promoted by high humidity, a condition mimicking the bag microenvironment. Germ tubes penetrated fruit via natural openings including stomata, lenticels, and surface cracks. To determine the chronology of infection by T. roseum, 'Fuji' fruit were inoculated in the field at different developmental stages. The earliest infection occurred 60 days after full bloom (dafb), and disease incidence increased as fruit maturity advanced. At harvest time (165 dafb), lesions on more recently inoculated fruit (105 dafb, 150 dafb) were larger than lesions from fruit inoculated on earlier dates. Histological observation showed that infection of younger fruit elicited stronger host lignification responses restricting lesion development. Taken together, our results support the hypothesis that high humidity in sealed bags and increased susceptibility associated with advancing fruit maturity are key factors promoting T. roseum infection and TBS symptom development on bagged apple fruit.