Complete genome sequence of a novel mitovirus isolated from the phytopathogenic fungus Alternaria alternata causing apple leaf blotch.

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata.

Monitoring studies of the occurrence of fire blight pathogen in Kazakhstan and identification of antagonistic microorganisms suppressing its development.

The paper presents data on phytosanitary monitoring of garden cenoses for fire blight in the Turkestan, Zhambyl, and Almaty regions of Kazakhstan. The purpose of this study is to assess the phytosanitary situation in various regions of Kazakhstan, determine the extent of fire blight spread, and isolate and identify the fire blight pathogen. During the study, methods such as hypersensitivity, pathogenicity, and fluorescent simplification-based specific hybridization polymerase chain reaction (FLASH-PCR) were used. It was found that in all the surveyed areas, disease foci were identified. For the first time, the fire blight pathogen was detected on fruit crops such as plum, peach, cherry plum, and quince, as well as on wild apricots. 274 plant samples were collected from which microorganisms were isolated. Isolates related to the fire blight pathogen Erwinia amylovora were identified by methods of hypersensitivity, pathogenicity, and FLASH-PCR diagnostics. Of the 156 isolates of microorganisms isolated from apple tree plant samples, 21 inhibited the in vitro growth of E. amylovora to varying degrees. Isolates 16.2 and 19.2 with maximum antagonistic activity were selected, where the pathogen growth inhibition zones were 52.2 ± 2.58 mm and 45.6 ± 0.55 mm, respectively. Based on the obtained sequence of nucleotides of the 16SpRNA gene site, it was found that the selected isolates with high antagonistic activity belonged to the Pseudomonas genus. In the future, based on these isolates, a new biological product for fire blight control can be created and adapted to the natural and climatic conditions of Kazakhstan.

Genome-Wide Identification and Expression Pattern Analysis of the WNK Gene Family in Apple under Abiotic Stress and Colletotrichum siamense Infection.

With-no-lysine kinase (WNK) is a unique serine/threonine kinase family member. WNK differs from other protein kinases by not having a standard lysine in subdomain II of the universally preserved kinase catalytic region. Conversely, the amino acid lysine located in subdomain I plays a crucial role in its phosphorylation. The WNK family has been reported to regulate Arabidopsis flowering, circadian rhythm, and abiotic stress. Eighteen members of the WNK gene family were discovered in apples in this research, and they were primarily grouped into five categories on the phylogenetic tree. Conserved domains and motifs also confirmed their identity as members of the WNK family. Promoter cis-acting element analysis indicated their potential role in responses to both abiotic stress and phytohormones. Furthermore, qRT-PCR analysis showed that the expression of MdWNK family genes was stimulated to different extents by Colletotrichum siamense, NaCl, mannitol, ABA, JA, and SA, with Colletotrichum siamense being the most prominent stimulant. MdWNK family genes were expressed across all apple tissues, with young fruits showing the greatest expression and roots showing the least expression. The research offered detailed insights into the MdWNK gene family, serving as a crucial basis for investigating the biological roles of MdWNK genes.

Gene function characterization in Aspergillus niger using a dual resistance marker transformation system mediated by Agrobacterium tumefaciens.

Aspergillus niger is a well-known workhorse for the industrial production of enzymes and organic acids. This fungus can also cause postharvest diseases in fruits. Although Agrobacterium tumefaciens-mediated transformation (ATMT) based on antibiotic resistance markers has been effectively exploited for inspecting functions of target genes in wild-type fungi, it still needs to be further improved in A. niger. In the present study, we re-examined the ATMT in the wild-type A. niger strains using the hygromycin resistance marker and introduced the nourseothricin resistance gene as a new selection marker for this fungus. Unexpectedly, our results revealed that the ATMT method using the resistance markers in A. niger led to numerous small colonies as false-positive transformants on transformation plates. Using the top agar overlay technique to restrict false positive colonies, a transformation efficiency of 87 ± 18 true transformants could be achieved for 106 conidia. With two different selection markers, we could perform both the deletion and complementation of a target gene in a single wild-type A. niger strain. Our results also indicated that two key regulatory genes (laeA and veA) of the velvet complex are required for A. niger to infect apple fruits. Notably, we demonstrated for the first time that a laeA homologous gene from the citrus postharvest pathogen Penicillium digitatum was able to restore the acidification ability and pathogenicity of the A. niger ΔlaeA mutant. The dual resistance marker ATMT system from our work represents an improved genetic tool for gene function characterization in A. niger.

Competition for nutrient niches within the apple blossom microbiota antagonizes the initiation of fire blight infection.

Changes in the plant microbiota composition are intimately associated with the health of the plant, but factors controlling the microbial community in flowers are poorly understood. In this study, we used apple flowers and fire blight as a model system to investigate the effects of floral microbiota and microbial competition on disease development and suppression. To compare changes in microbial flora with the RNA expression patterns of plants, the flower samples were collected in three different flowering stages (Bud, Popcorn, and Full-bloom). Using advanced sequencing technology, we analyzed the data and conducted both in vitro and in vivo experiments to validate our findings. Our results show that the Erwinia amylovora use arabinogalactan, which is secreted on the flowers, for early colonization of apple flowers. Pantoea agglomerans was more competitive for arabinogalactan than E. amylovora. Additionally, P. agglomerans suppressed the expression of virulence factors of E. amylovora by using arabinose, which is a major component of arabinogalactan, which induces virulence gene expression. The present data provide new insights into developing control strategies for diverse plant diseases, including fire blight, by highlighting the importance of nutrients in disease development or suppression.

Comparative analysis on natural variants of fire blight resistance protein FB_MR5 indicates distinct effector recognition mechanisms.

FB_MR5 is a nucleotide-binding domain and leucine-rich repeat protein identified from wild apple species Malus × robusta 5 conferring disease resistance to bacterial fire blight. FB_MR5 (hereafter MrMR5) recognizes the cysteine protease effector EaAvrRpt2 secreted from the causal agent of bacterial fire blight, Erwinia amylovora. We previously reported that MrMR5 is activated by the C-terminal cleavage product (ACP3) of Malus domestica RIN4 (MdRIN4) produced by EaAvrRpt2-directed proteolysis. We show that MbMR5 from a wild apple species Malus baccata shares 99.4% amino acid sequence identity with MrMR5. Surprisingly, transient expression of MbMR5 in Nicotiana benthamiana showed autoactivity in contrast to MrMR5. Domain swap and mutational analyses revealed that 1 amino acid polymorphism in the MbMR5 CC domain is critical in enhancing autoactivity. We further demonstrated that MrMR5 carrying 7 amino acid polymorphisms present in MbMR5 is not activated by MdRIN4 ACP3 but recognizes AvrRpt2 without MdRIN4 in N. benthamiana. Our findings indicate that naturally occurring polymorphisms of MR5 natural variants can confer its cell death-inducing activity and the effector recognition mechanism likely due to altered compatibility with RIN4.

Deciphering the mechanisms involved in reduced sensitivity to azoles and fengycin lipopeptide in Venturia inaequalis.

Apple scab, caused by the hemibiotrophic fungus Venturia inaequalis, is currently the most common and damaging disease in apple orchards. Two strains of V. inaequalis (S755 and Rs552) with different sensitivities to azole fungicides and the bacterial metabolite fengycin were compared to determine the mechanisms responsible for these differences. Antifungal activity tests showed that Rs552 had reduced sensitivity to tebuconazole and tetraconazole, as well as to fengycin alone or in a binary mixture with other lipopeptides (iturin A, pumilacidin, lichenysin). S755 was highly sensitive to fengycin, whose activity was close to that of tebuconazole. Unlike fengycin, lipopeptides from the iturin family (mycosubtilin, iturin A) had similar activity on both strains, while those from the surfactin family (lichenysin, pumilacidin) were not active, except in binary mixtures with fengycin. The activity of lipopeptides varies according to their family and structure. Analyses to determine the difference in sensitivity to azoles (which target the CYP51 enzyme involved in the ergosterol biosynthesis pathway) showed that the reduced sensitivity in Rs552 is linked to (i) a constitutive increased expression of the Cyp51A gene caused by insertions in the upstream region and (ii) greater efflux by membrane pumps with the involvement of ABC transporters. Microscopic observations revealed that fengycin, known to interact with plasma membranes, induced morphological and cytological changes in cells from both strains. Sterol and phospholipid analyses showed a higher level of ergosta-7,22-dien-3-ol and a lower level of PI(C16:0/C18:1) in Rs552 compared with S755. These differences could therefore influence the composition of the plasma membrane and explain the differential sensitivity of the strains to fengycin. However, the similar antifungal activities of mycosubtilin and iturin A in the two strains indirectly indicate that sterols are probably not involved in the fengycin resistance mechanism. This leads to the conclusion that different mechanisms are responsible for the difference in susceptibility to azoles or fengycin in the strains studied.

Identification and evaluation of an endophytic antagonistic yeast for the control of gray mold (Botrytis cinerea) in apple and mechanisms of action.

Gray mold, caused by Botrytis cinerea, is a prevalent postharvest disease of apple that limits their shelf life, resulting in significant economic losses. The use of antagonistic microorganisms has been shown to be an effective approach for managing postharvest diseases of fruit. In the present study, an endophytic yeast strain PGY-2 was isolated from apples and evaluated for its biocontrol efficacy against gray mold and its mechanisms of action. Results indicated that strain PGY-2, identified as Bullera alba, reduced the occurrence of gray mold on apples and significantly inhibited lesion development in pathogen-inoculated wounds. Gray mold control increased with the use of increasing concentrations of PGY-2, with the best disease control observed at 108 cells/mL. Notably, Bullera alba PGY-2 did not inhibit the growth of Botrytis cinerea in vitro indicating that the yeast antagonist did not produce antimicrobial compounds. The rapid colonization and stable population of PGY-2 in apple wounds at 4 °C and 25 °C confirmed its ability to compete with pathogens for nutrients and space. PGY-2 also had a strong ability to form a biofilm and enhanced the activity of multiple defense-related enzymes (POD, PPO, APX, SOD, PAL) in host tissues. Our study is the first time to report the use of Bullera alba PGY-2 as a biocontrol agent for postharvest diseases of apple and provide evidence that Bullera alba PGY-2 represents an endophytic antagonistic yeast with promising biocontrol potential and alternative to the use of synthetic, chemical fungicides for the control of postharvest gray mold in apples.

Bioactivities evaluation of an endophytic bacterial strain Bacillus tequilensis QNF2 inhibiting apple ring rot caused by Botryosphaeria dothidea on postharvest apple fruits.

Apple ring rot, one of the most common apple postharvest diseases during storage, is caused by Botryosphaeria dothidea. Presently, the disease management is primarily dependent on chemical fungicide application. Here we demonstrated an endophyte bacterium Bacillus tequilensis QNF2, isolated from Chinese leek (Allium tuberosum) roots considerably suppressed B. dothidea mycelial growth, with the highest suppression of 73.56 % and 99.5 % in the PDA and PDB medium, respectively in vitro confront experiments. In in vivo experiments, B. tequilensis QNF2 exhibited a control efficacy of 88.52 % and 100 % on ring rot disease on postharvest apple fruits inoculated with B. dothidea disc and dipped into B. dothidea culture, respectively. In addition, B. tequilensis QNF2 volatile organic compounds (VOCs) also manifested markedly inhibition against B. dothidea mycelial growth and the ring rot on postharvest apple fruits. Moreover, B. tequilensis QNF2 severely damaged the mycelial morphology of B. dothidea. Finally, B. tequilensis QNF2 significantly repressed the expression of six pathogenicity-related genes, such as adh, aldh, aldh3, galm, pdc1, pdc2, involved in glycolysis/gluconeogenesis of B. dothidea. The findings of the study proved that B. tequilensis QNF2 was a promising alternative for controlling apple ring rot of postharvest apple fruit.

Application of Environmental Monitoring Programs and Root Cause Analysis to Identify and Implement Interventions to Reduce or Eliminate Listeria Populations in Apple Packinghouses.

Controlling Listeria in produce packinghouses can be challenging due to the large number of potential contamination routes. For example, repeated isolation of the same Listeria subtype in a packinghouse could indicate persistence in the packinghouse or reintroduction of the same Listeria from an upstream source. To improve understanding of Listeria transmission patterns in packinghouses, we performed a longitudinal study in four apple packinghouses, including testing of 1,339 environmental sponges and whole genome sequencing (WGS)-based characterization of 280 isolates. Root cause analysis and subsequent intervention implementation were also performed and assessed for effectiveness. Listeria prevalence among environmental sponges collected from the four packinghouses was 20% (range of 5-31% for individual packinghouses). Sites that showed high Listeria prevalence included drains, forklift tires and forks, forklift stops, and waxing area equipment frames. A total of 240/280 WGS-characterized isolates were represented in 41 clusters, each containing two or more isolates that differed by ≤50 high-quality single nucleotide polymorphisms (hqSNPs); 21 clusters were isolated from one packinghouse over ≥2 samplings (suggesting persistence or possibly reintroduction), while 11 clusters included isolates from >2 packinghouses, suggesting common upstream sources. Some interventions successfully (i) reduced Listeria detection on forklift tires and forks (across packinghouses) and (ii) mitigated packinghouse-specific Listeria issues (e.g., in catch pans). However, interventions that lacked enhanced equipment disassembly when persistence was suspected typically appeared to be unsuccessful. Overall, while our data suggest a combination of intensive environmental sampling with subtyping and root cause analysis can help identify effective interventions, implementation of effective interventions continues to be a challenge in packinghouses.

Synthesis of aspartic acid and tyrosine by the fire blight pathogen Erwinia amylovora is not required for proliferation on apple flower stigmas or virulence in fruitlets.

AIMS: The Gram-negative bacterium Erwinia amylovora (Ea) is the causal agent of fire blight, a devastating disease of apples and pears. In the fire blight disease cycle, Ea grows in different plant tissues, each presenting a distinct nutrient environment. Here, we investigate the ability of aspartate and tyrosine double auxotroph Ea lines to proliferate on apple flower stigma surfaces representing the epiphytic growth stage of Ea and in developing fruitlets representing one endophytic growth stage of Ea. METHODS AND RESULTS: Heterologous complementation studies in an Escherichia coli aspartate and tyrosine auxotroph verify that Ea aspartate aminotransferase (AspC) and tyrosine aminotransferase (TyrB) act as aspartate and tyrosine amino transferases. Growth analysis reveals that Ea aspC tyrB mutants multiply to near-wild-type levels on apple flower stigmas and immature fruitlets. CONCLUSIONS: Ea AspC and TyrB are reciprocally complementing for aspartate and tyrosine synthesis in Ec and in Ea. Ea aspC  and  tyrB mutants obtain sufficient aspartate and tyrosine to support multiplication on stigma surfaces and virulence in immature fruitlets.

A non-invasive method for phenotyping scab-tolerant apple plants using volatile organic compounds.

One of the most devastating diseases of apples is scab, caused by the fungus Venturia inaequalis. Most commercial apple varieties are susceptible to this disease; only a few are resistant. Breeding approaches are being used to develop better apple varieties that are resistant to scab. Volatile organic compounds (VOCs) contribute greatly to a plant's phenotype, and their emission profile largely depends on the genotype. In the non-destructive phenotyping of plants, VOCs can be used as biomarkers. In this study, we assessed non-destructively the scab tolerance potential of resistant (cv. 'Prima') and susceptible (cv. 'Oregon Spur') apple cultivars by comparing their major leaf VOC compositions and relative proportions. A comparison of the leaf VOC profiles of the two cultivars revealed 16 different VOCs, with cis-3-hexenyl acetate (3HA) emerging as a biomarker of cultivar differences. V. inaequalis growth was significantly inhibited in vitro by 3HA treatment. 3HA was significantly effective in reducing scab symptoms on V. inaequalis-inoculated leaves of 'Oregon Spur.' The resistant cultivar 'Prima' also exhibited higher lipoxygenase (LOX) activity and α-linolenic acid (ALA) levels, suggesting that V. inaequalis resistance is linked to LOX activity and 3HA biosynthesis. This study proposes 3HA as a potential biomarker for rapid non-destructive screening of scab-resistant apple germplasm of 'Prima' based on leaf VOCs.

Plant growth promotion and biocontrol properties of a synthetic community in the control of apple disease.

BACKGROUND: Apple Replant Disease (ARD) is common in major apple-growing regions worldwide, but the role of rhizosphere microbiota in conferring ARD resistance and promoting plant growth remains unclear. RESULTS: In this study, a synthetic microbial community (SynCom) was developed to enhance apple plant growth and combat apple pathogens. Eight unique bacteria selected via microbial culture were used to construct the antagonistic synthetic community, which was then inoculated into apple seedlings in greenhouse experiments. Changes in the rhizomicroflora and the growth of aboveground plants were monitored. The eight strains, belonging to the genera Bacillus and Streptomyces, have the ability to antagonize pathogens such as Fusarium oxysporum, Rhizoctonia solani, Botryosphaeria ribis, and Physalospora piricola. Additionally, these eight strains can stably colonize in apple rhizosphere and some of them can produce siderophores, ACC deaminase, and IAA. Greenhouse experiments with Malus hupehensis Rehd indicated that SynCom promotes plant growth (5.23%) and increases the nutrient content of the soil, including soil organic matter (9.25%) and available K (1.99%), P (7.89%), and N (0.19%), and increases bacterial richness and the relative abundance of potentially beneficial bacteria. SynCom also increased the stability of the rhizosphere microbial community, the assembly of which was dominated by deterministic processes (|ß NTI| > 2). CONCLUSIONS: Our results provide insights into the contribution of the microbiome to pathogen inhibition and host growth. The formulation and manipulation of similar SynComs may be a beneficial strategy for promoting plant growth and controlling soil-borne disease.

Unravelling the impact of environmental factors in shaping plant microbiomes.

This article emphasizes the significant role of environmental factors in shaping the plant microbiome, highlighting how bacterial and fungal communities influence plant responses to water stress, and how environmental factors shape fungal communities in crops. Furthermore, recent studies describe how different genotypes and levels of water stress affect the composition of bacterial communities associated with quinoa plants, as well as the relationship between environmental factors and the structure of fungal communities in apple fruit. These findings underscore the importance of understanding plant microbiome dynamics in developing effective crop protection strategies and improving agricultural sustainability with the objective of advance towards a microbiome-based strategy which allows us to improve crop tolerance to abiotic stresses.

Fungal invasion-induced accumulation of salicylic acid promotes anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple fruits.

In the field, necrosis area induced by pathogens is usually surrounded by a red circle in apple fruits. However, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we demonstrated that accumulated salicylic acid (SA) induced by fungal infection promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple (Malus domestica). Inoculating apple fruits with Valsa mali or Botryosphaeria dothidea induced a red circle surrounding the necrosis area, which mimicked the phenotype observed in the field. The red circle accumulated a high level of anthocyanins, which was positively correlated with SA accumulation stimulated by fungal invasion. Further analysis showed that SA promoted anthocyanin biosynthesis in a dose-dependent manner in both apple calli and fruits. We next demonstrated that MdNPR1, a master regulator of SA signaling, positively regulated anthocyanin biosynthesis in both apple and Arabidopsis. Moreover, MdNPR1 functioned as a co-activator to interact with and enhance the transactivation activity of MdTGA2.2, which could directly bind to the promoters of anthocyanin biosynthetic and regulatory genes to promote their transcription. Suppressing expression of either MdNPR1 or MdTGA2.2 inhibited coloration of apple fruits, while overexpressing either of them significantly promoted fruit coloration. Finally, we revealed that silencing either MdNPR1 or MdTGA2.2 in apple fruits repressed SA-induced fruit coloration. Therefore, our data determined that fungal-induced SA promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module, resulting in a red circle surrounding the necrosis area in apple fruits.

Bacillus B2 promotes root growth and enhances phosphorus absorption in apple rootstocks by affecting MhMYB15.

Due to the chelation of phosphorus in the soil, it becomes unavailable for plant growth and development. The mechanisms by which phosphorus-solubilizing bacteria activate immobilized phosphorus to promote the growth and development of woody plants, as well as the intrinsic molecular mechanisms, are not clear. Through the analysis of microbial communities in the rhizosphere 16S V3-V4 and a homologous gene encoding microbial alkaline phosphomonoesterase (phoD) in phosphate-efficient (PE) and phosphate-inefficient apple rootstocks, it was found that PE significantly enriched beneficial rhizobacteria. The best phosphorus-solubilizing bacteria, Bacillus sp. strain 7DB1 (B2), was isolated, purified, and identified from the rhizosphere soil of PE rootstocks. Incubating with Bacillus B2 into the rhizosphere of apple rootstocks significantly increased the soluble phosphorus and flavonoid content in the rhizosphere soil. Simultaneously, this process stimulates the root development of the rootstocks and enhances plant phosphorus uptake. After root transcriptome sequencing, candidate transcription factor MhMYB15, responsive to Bacillus B2, was identified through heatmap and co-expression network analysis. Yeast one-hybrid, electrophoretic mobility shift assay, and LUC assay confirmed that MhMYB15 can directly bind to the promoter regions of downstream functional genes, including chalcone synthase MhCHS2 and phosphate transporter MhPHT1;15. Transgenic experiments with MhMYB15 revealed that RNAi-MhMYB15 silenced lines failed to induce an increase in flavonoid content and phosphorus levels in the roots under the treatment of Bacillus B2, and plant growth was slower than the control. In conclusion, MhMYB15 actively responds to Bacillus B2, regulating the accumulation of flavonoids and the uptake of phosphorus, thereby influencing plant growth and development.

Unveiling the fungal diversity and associated secondary metabolism on black apples.

Black apples are the result of late-stage microbial decomposition after falling to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum. Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by the cultivation of 15 recently isolated fungal strains on 9 different substrates in a One Strain Many Compounds (OSMAC) approach, leading to the identification of 3,319 unique chemical features. Only 6.4% were attributable to known compounds based on analysis of high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS/MS) data using spectral library matching tools. Of the 1,606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training data set. Notably, the detection of several antifungal compounds indicates the importance of such compounds for the invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples were found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena.IMPORTANCEMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavors. Hence, the ecological role of such microbial secondary metabolites must be considered to understand how best to utilize such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit," with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes, and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes.

Endophytic fungal isolates from apple tissue: Latent pathogens lurking within?

Fungal endophytes inhabit a similar ecological niche to that occupied by many phytopathogens, with several pathogens isolated from healthy tissues in their latent phase. This study aimed to evaluate the pathogenicity, the colonisation ability, and the enzyme activity of 37 endophytic fungal isolates recovered from apparently healthy apple shoot and leaf tissues. The pathogenicity of the isolates was assessed on 'Royal Gala' and 'Braeburn' fruit and detached 'Royal Gala' shoots. For the non-pathogenic isolates, their ability to endophytically colonise detached 'Royal Gala' shoots was evaluated. Enzyme activity assays were undertaken to determine whether the pathogenicity of the endophytes was related to the production of the extracellular enzymes, amylase, cellulase, pectinase, protease, and xylanase. Of the 37 isolates studied, eight isolates, representing the genera Colletotrichum, Diaporthe, Fusarium, and Penicillium, were shown to be pathogenic on both apple shoots and fruit. Two isolates identified as Trichoderma atroviride, were pathogenic only on shoots, and three isolates, representing the genus Diaporthe, were pathogenic only on fruit. Of the remaining 24 isolates, 22 (Biscogniauxia (n = 8), Chaetomium (n = 4), Trichoderma (n = 3), Epicoccum (n = 2), Neosetophoma (n = 2), Xylaria (n = 1), Daldinia (n = 1), and Paraphaeosphaeria (n = 1)) were recovered from the inoculated apple shoots but two failed to colonise the shoot tissues. Of the isolates tested, 20 produced amylase, 15 cellulase, 25 pectinase, 26 protease, and 13 xylanase. There was no correlation between the range and type of enzymes produced by the isolates and their pathogenicity or ability to endophytically colonise the shoot tissue. The study showed that approximately one-third (13/37) of the isolates recovered from the apparently healthy apple shoot tissues were observed as latent pathogens. The isolates that did not cause disease symptoms may have the ability to reduce colonisation of apple tissues by pathogens including Neonectria ditissima associated with European canker of apple.

Detecting fungi-affected multi-crop disease on heterogeneous region dataset using modified ResNeXt approach.

Crop diseases pose significant threats to agriculture, impacting crop production. Biotic factors contribute to various diseases, including fungal, bacterial, and viral infections. Recent advancements in deep learning present a novel approach to the detection and recognition of these crop diseases. While considerable research has focused on identifying and recognizing crop diseases, fungal disease-affected crops have received relatively less attention and also detecting disease on different region datasets. This paper is about spotting fungal diseases in crops across different regions with diverse climates. It emphasizes the need for tailored detection methods, addressing the risk of mycotoxin production by fungi, which can harm both humans and animals. Detecting fungal diseases in apple, guava, and custard apple crops such as spot, scab, rust, rot, leaf spot, and insect ate. In the proposed work, the modified ResNeXt variant of the convolution neural network (CNN) technique was employed to predict 3 major crop classes of fungal disease. Initially, using Inception-v7 and ResNet for fungal disease in crops did not yield satisfactory results. A modified ResNeXt CNN model was proposed, showing improved fungal disease prediction. The novel model underwent a comparison with established methodologies. The suggested model draws upon a benchmark dataset consisting of 14,408 images capturing fungal diseases, categorized into three distinct classes: apple, custard apple, and guava. Experimental outcomes show that the proposed mutated ResNeXt model outperformed the state-of-the-art approaches. The model achieved 98.92% accuracy and high performance across recall, precision, and F1-score (above 99%) for the benchmark dataset, which gained encouragement and was comparable with the state-of-the-art approach.

VmRDR2 of Valsa mali mediates the generation of VmR2-siR1 that suppresses apple resistance by RNA interference.

Cross-kingdom RNA interference (RNAi) is a crucial mechanism in host-pathogen interactions, with RNA-dependent RNA polymerase (RdRP) playing a vital role in signal amplification during RNAi. However, the role of pathogenic fungal RdRP in siRNAs generation and the regulation of plant-pathogen interactions remains elusive. Using deep sequencing, molecular, genetic, and biochemical approaches, this study revealed that VmRDR2 of Valsa mali regulates VmR2-siR1 to suppress the disease resistance-related gene MdLRP14 in apple. Both VmRDR1 and VmRDR2 are essential for the pathogenicity of V. mali in apple, with VmRDR2 mediating the generation of endogenous siRNAs, including an infection-related siRNA, VmR2-siR1. This siRNA specifically degrades the apple intracellular LRR-RI protein gene MdLRP14 in a sequence-specific manner, and overexpression of MdLRP14 enhances apple resistance against V. mali, which can be suppressed by VmR2-siR1. Conversely, MdLRP14 knockdown reduces resistance. In summary, this study demonstrates that VmRDR2 contributes to the generation of VmR2-siR1, which silences the host's intracellular LRR protein gene, thereby inhibiting host resistance. These findings offer novel insights into the fungi-mediated pathogenicity mechanism through RNAi.