Alternaria solani effectors AsCEP19 and AsCEP20 reveal novel functions in pathogenicity and conidiogenesis.

Previous work identified a pair of specific effectors AsCEP19 and AsCEP20 in Alternaria solani as contributors to the virulence of A. solani. Here, we constructed AsCEP19 and AsCEP20 deletion mutants in A. solani strain HWC168 to further reveal the effects of these genes on the biology and pathogenicity of A. solani. Deletion of AsCEP19 and AsCEP20 did not affect vegetative growth but did affect conidial maturation, with an increase in the percentage of abnormal conidia produced. Furthermore, we determined the expression patterns of genes involved in the conidiogenesis pathway and found that the regulatory gene abaA was significantly upregulated and chsA, a positive regulator for conidiation, was significantly downregulated in the mutant strains compared to the wild-type strain. These results suggest that AsCEP19 and AsCEP20 indirectly affect the conidial development and maturation of A. solani. Pathogenicity assays revealed significantly impaired virulence of ΔAsCEP19, ΔAsCEP20, and ΔAsCEP19 + AsCEP20 mutants on potato and tomato plants. Moreover, we performed localization assays with green fluorescent protein-tagged proteins in chili pepper leaves. We found that AsCEP19 can specifically localize to the chloroplasts of chili pepper epidermal cells, while AsCEP20 can localize to both chloroplasts and the plasma membrane. Weighted gene co-expression network analysis revealed enrichment of genes of this module in the photosynthesis pathway, with many hub genes associated with chloroplast structure and photosynthesis. These results suggest that chloroplasts are the targets for AsCEP19 and AsCEP20. IMPORTANCE: Alternaria solani is an important necrotrophic pathogen causing potato early blight. Previous studies have provide preliminary evidence that specific effectors AsCEP19 and AsCEP20 contribute to virulence, but their respective functions, localization, and pathogenic mechanisms during the infection process of A. solani remain unclear. Here, we have systematically studied the specific effectors AsCEP19 and AsCEP20 for the first time, which are essential for conidial maturation. The deletion of AsCEP19 and AsCEP20 can significantly impair fungal pathogenicity. Additionally, we preliminarily revealed that AsCEP19 and AsCEP20 target the chloroplasts of host cells. Our findings further enhance our understanding of the molecular mechanisms underlying the virulence of necrotrophic pathogens.

Transcriptome analysis reveals the humic acids and chitosan suppressing Alternaria solani growth.

AIMS: Understanding the inhibitory effects of natural organic substances on soil-borne pathogenic fungi and the relevant molecular mechanisms are highly important for future development of green prevention and control technology against soil-borne diseases. Our study elucidates the inhibitory effect of the combined application of humic acids (HAs) and chitosan on Alternariasolani and the light on the corresponding mechanism. METHODS AND RESULTS: The effect on A. solani growth by HAs incorporated with chitosan was investigated by plate culture and the corresponding mechanism was revealed using transcriptomics. The colony growth of A. solani was suppressed with the highest inhibition rate 33.33% when swine manure HAs was compounded with chitosan at a ratio of 1:4. Chitosan changed the colony morphology from round to irregularly. RNA-seq in the HAs and chitosan (HC) treatment revealed 239 differentially expressed genes compared with the control. The unigenes associated with enzymes activities related to growth and biological processes closely related to mycelial growth and metabolism were downregulated. RNA-seq also revealed that chitosan altered the expression of genes related to secondary metabolism, fungal cell wall formation and polysaccharide synthesis, and metabolism. Meanwhile, weighted gene co-expression network analysis showed that, genes expression in the module positively correlated with mycelial growth was significantly reduced in the HC treatment; and the results were verified by real-time quantitative polymerase chain reaction. CONCLUSIONS: The co-inhibition effect of HAs and chitosan on A. solani is associated with downregulated genes expression correlated with mycelial growth.

Green-fabricated silver nanoparticles from Quercus incana leaf extract to control the early blight of tomatoes caused by Alternaria solani.

BACKGROUND: Early blight (EB) of Tomatoes, caused by Alternaria solani, is a serious fungal disease that adversely affects tomato production. Infection is characterized by dark lesions on leaves, stems, and fruits. Several agrochemicals can be used to control infection, these chemicals may disrupt environmental equilibrium. An alternative technology is needed to address this significant fungal threat. This study was designed to control the growth of EB in tomatoes caused by A. solani, using green-fabricated silver nanoparticles (Ag-NPs). RESULTS: Ag-NPs were synthesized through an environmentally friendly and cost-effective approach using leaf extract of Quercus incana Roxb. (Fagaceae). The physico-chemical characterization of the Ag-NPs was conducted through UV-visible spectroscopy, scanning electron microscopy, X-ray diffraction analysis, and Fourier transform infrared spectrometry. The Ag-NPs produced were round with a mean diameter of 27 nm. The antifungal activity of these Ag-NPs was assessed through in vitro Petri plate and in vitro leaflet assays against A. solani. The green fabricated Ag-NPs exhibited excellent antifungal activity in vitro at a concentration of 100 mg/l against A. solani, inhibiting growth by 98.27 ± 1.58% and 92.79 ± 1.33% during Petri plate and leaflet assays, respectively. CONCLUSION: In conclusion, this study suggests the practical application of green-fabricated Ag-NPs from Q. incana leaf extract against A. solani to effectively control EB disease in tomatoes.

Exploring the mechanism underlying the antifungal activity of chitosan-based ZnO, CuO, and SiO2 nanocomposites as nanopesticides against Fusarium solani and Alternaria solani.

Chitosan-based nanocomposites (CS NCs) are gaining considerable attention as multifaceted antifungal agents. This study investigated the antifungal activity of NCs against two phytopathogenic strains: Fusarium solani (F. solani) and Alternaria solani (A. solani). Moreover, it sheds light on their underlying mechanisms of action. The NCs, CS-ZnO, CS-CuO, and CS-SiO2, were characterized using advanced methods. Dynamic and electrophoretic light scattering techniques revealed their size range (60-170 nm) and cationic nature, as indicated by the positive zeta potential values (from +16 to +22 mV). Transmission electron microscopy revealed the morphology of the NCs as agglomerates formed between the chitosan and oxide components. X-ray diffraction patterns confirmed crystalline structures with specific peaks indicating their constituents. Antifungal assessments using the agar diffusion technique demonstrated significant inhibitory effects of the NCs on both fungal strains (1.5 to 4-fold), surpassing the performance of the positive control, nystatin. Notably, the NCs exhibited superior antifungal potency, with CS-ZnO NCs being the most effective. A. solani was the most sensitive strain to the studied agents. Furthermore, the tested NCs induced oxidative stress in fungal cells, which elevated stress biomarker levels, such as superoxide dismutase (SOD) activity and protein carbonyl content (PCC), 2.5 and 6-fold for the most active CS-CuO in F. solani respectively. Additionally, they triggered membrane lipid peroxidation up to 3-fold higher compared to control, a process that potentially compromises membrane integrity. Laurdan fluorescence spectroscopy highlighted alterations in the molecular organization of fungal cell membranes induced by the NCs. CS-CuO NCs induced a membrane rigidifying effect, while CS-SiO2 and CS-ZnO could rigidify membranes in A. solani and fluidize them in F. solani. In summary, this study provides an in-depth understanding of the interactions of CS-based NCs with two fungal strains, showing their antifungal activity and offering insights into their mechanisms of action. These findings emphasize the potential of these NCs as effective and versatile antifungal agents.

Ultra-high-resolution UAV-imaging and supervised deep learning for accurate detection of Alternaria solani in potato fields.

Alternaria solani is the second most devastating foliar pathogen of potato crops worldwide, causing premature defoliation of the plants. This disease is currently prevented through the regular application of detrimental crop protection products and is guided by early warnings based on weather predictions and visual observations by farmers. To reduce the use of crop protection products, without additional production losses, it would be beneficial to be able to automatically detect Alternaria solani in potato fields. In recent years, the potential of deep learning in precision agriculture is receiving increasing research attention. Convolutional Neural Networks (CNNs) are currently the state of the art, but also come with challenges, especially regarding in-field robustness. This stems from the fact that they are often trained on datasets that are limited in size or have been recorded in controlled environments, not necessarily representative of real-world settings. We collected a dataset consisting of ultra-high-resolution modified RGB UAV-imagery of both symptomatic and non-symptomatic potato crops in the field during various years and disease stages to cover the great variability in agricultural data. We developed a convolutional neural network to perform in-field detection of Alternaria, defined as a binary classification problem. Our model achieves a similar accuracy as several state-of-the-art models for disease detection, but has a much lower inference time, which enhances its practical applicability. By using training data of three consecutive growing seasons (2019, 2020 and 2021) and test data of an independent fourth year (2022), an F1 score of 0.93 is achieved. Furthermore, we evaluate how different properties of the dataset such as its size and class imbalance impact the obtained accuracy.

Multi-omics approaches to understand pathogenicity during potato early blight disease caused by Alternaria solani.

Potato early blight (PEB), a foliar disease of potato during the growing period, caused by Alternaria sp., is common in major potato-producing areas worldwide. Effective agents to control this disease or completely resistant potato varieties are absent. Large-scale use of fungicides is limited due to possibility of increase in pathogen resistance and the requirements of ecological agriculture. In this study, we focused on the composition and infection characteristics of early blight pathogens in Yunnan Province and screened candidate pathogenesis-related pathways and genes. We isolated 85 strains of Alternaria sp. fungi from typical early blight spots in three potato-growing regions in Yunnan Province from 2018 to 2022, and identified 35 strains of Alternaria solani and 50 strains of Alternaria alternata by morphological characterization and ITS sequence comparison, which were identified as the main and conditional pathogens causing early blight in potato, respectively. Scanning electron microscope analysis confirmed only A. solani producing appressorium at 4 h after inoculation successfully infected the leaf cells. Via genome assembly and annotation, combine transcriptome and proteomic analysis, the following pathogenicity-related unit, transcription factors and metabolic pathway were identified: (1) cell wall-degrading enzymes, such as pectinase, keratinase, and cellulase; (2) genes and pathways related to conidia germination and pathogenicity, such as ubiquitination and peroxisomes; and (3) transcription factors, such as Zn-clus, C2H2, bZIP, and bHLH. These elements were responsible for PEB epidemic in Yunnan.

Use of ginger extract and bacterial inoculants for the suppression of Alternaria solani causing early blight disease in Tomato.

Early blight (EB), caused by Alternaria solani, is a serious problem in tomato production. Plant growth-promoting rhizobacteria promote plant growth and inhibit plant disease. The present study explored the bio-efficacy of synergistic effect of rhizobacterial isolates and ginger powder extract (GPE) against tomato EB disease, singly and in combination. Six fungal isolates from symptomatic tomato plants were identified as A. solani on the basis of morphological features i.e., horizontal septation (6.96 to 7.93 µm), vertical septation (1.50 to 2.22 µm), conidia length (174.2 to 187.6 µm), conidial width (14.09 to 16.52 µm), beak length (93.06 to 102.26 µm), and sporulation. Five of the twenty-three bacterial isolates recovered from tomato rhizosphere soil were nonpathogenic to tomato seedlings and were compatible with each other and with GPE. Out of five isolates tested individually, three isolates (St-149D, Hyd-13Z, and Gb-T23) showed maximum inhibition (56.3%, 48.3%, and 42.0% respectively) against mycelial growth of A. solani. Among combinations, St-149D + GPE had the highest mycelial growth inhibition (76.9%) over the untreated control. Bacterial strains molecularly characterized as Pseudomonas putida, Bacillus subtilis, and Bacillus cereus and were further tested in pot trials through seed bacterization for disease control. Seeds treated with bacterial consortia + GPE had the highest disease suppression percentage (78.1%), followed by St-149D + GPE (72.2%) and Hyd-13Z + GPE (67.5%). Maximum seed germination was obtained in the bacterial consortia + GPE (95.0 ± 2.04) followed by St-149D + GPE (92.5 ± 1.44) and Hyd-13Z + GPE (90.0 ± 2.04) over control (73.8 ± 2.39) and chemical control as standard treatment (90.0 ± 2). Ginger powder extracts also induce the activation of defence-related enzymes (TPC, PO, PPO, PAL, and CAT) activity in tomato plants. These were highly significant in the testing bacterial inoculants against A. solani infection in tomato crops.

Positive selection and functional diversification of transcription factor Cmr1 homologs in Alternaria.

Transcription factor Cmr1 (Colletotrichum melanin regulation 1) and its homologs in several plant fungal pathogens are the regulators of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis pathway and have evolved functional diversification in morphology and pathogenicity. The fungal genus Alternaria comprises the group of "black fungi" that are rich in DHN-melanin in the primary cell wall and septa of the conidia. Some Alternaria species cause many economically important plant diseases worldwide. However, the evolution and function of Cmr1 homologs in Alternaria remain poorly understood. Here, we identified a total of forty-two Cmr1 homologs from forty-two Alternaria spp. and all contained one additional diverse fungal specific transcription factor motif. Phylogenetic analysis indicated the division of these homologs into five major clades and three branches. Dated phylogeny showed the A and D clades diverged latest and earliest, respectively. Molecular evolutionary analyses revealed that three amino acid sites of Cmr1 homologs in Alternaria were the targets of positive selection. Asmr1, the homolog of Cmr1 in the potato early blight pathogen, Alternaria solani was amplified and displayed the sequence conservation at the amino acid level in different A. solani isolates. Asmr1 was further confirmed to have the transcriptional activation activity and was upregulated during the early stage of potato infection. Deletion of asmr1 led to the decreased melanin content and pathogenicity, deformed conidial morphology, and responses to cell wall and fungicide stresses in A. solani. These results suggest positive selection and functional divergence have played a role in the evolution of Cmr1 homologs in Alternaria. KEY POINTS: • Cmr1 homologs were under positive selection in Alternaria species • Asmr1 is a functional transcription factor, involved in spore development, melanin biosynthesis, pathogenicity, and responses to cell wall and fungicide stresses in A. solani • Cmr1 might be used as a potential taxonomic marker of the genus Alternaria.

First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 µm, and 80.7 × 3.9 µm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.

Biological control of tomato early blight in Pakistan using local rhizobacteria.

BACKGROUND: The biocontrol potential of soil microbes can reduce the extensive use of hazardous synthetic fungicides. This study was designed to find a strain of rhizobacteria indigenous to Pakistan with potential biocontrol against early blight of tomato caused by Alternaria solani and to characterize its biocontrol mechanisms. RESULTS: Among 88 strains tested for antagonism against A. solani on agar media, S27, Dt10 and 423, identified by 16S rRNA sequencing as strains of Bacillus amyloliquefaciens, B. cereus and Stenotrophomonas rhizophila, respectively, were the most inhibitory. When applied to detached tomato leaflets in Petri dish assays, the strains reduced lesion development by over 30% compared to the control. In greenhouse pot trials, the bacterial strains reduced early blight severity by over 50%. In three field trials, all three strains applied to tomato foliage slowed early blight disease progress and reduced disease severity, with B. amyloliquefaciens S27 reducing the area under the disease progress curve by up to 70%. All three strains showed protease, catalase and oxidase activities in vitro, but none produced ß-1,3-glucanase and only B. cereus Dt10 showed slight chitinase activity. In a greenhouse experiment in which the bacteria were applied to tomato foliage prior to pathogen inoculation, bacteria-treated leaves had higher ß-1,3-glucanase and chitinase levels than leaves inoculated only with the pathogen, indicating priming induction of response. CONCLUSION: Three rhizobacteria strains have the potential to control early blight of tomato under Pakistan's growing conditions, with B. amyloliquefaciens S27 being the most promising candidate for commercial development. Antagonism and induction of the priming response may be mechanisms of biocontrol by the bacterial strains. © 2023 Society of Chemical Industry.

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

Microbial consortium mediated acceleration of the defense response in potato against Alternaria solani through prodigious inflation in phenylpropanoid derivatives and redox homeostasis.

The present study was carried out in a pot experiment to examine the bioefficacy of three biocontrol agents, viz., Trichoderma viride, Bacillus subtilis, and Pseudomonas fluorescens, either alone or in consortium, on plant growth promotion and activation of defense responses in potato against the early blight pathogen Alternaria solani. The results demonstrate significant enhancement in growth parameters in plants bioprimed with the triple-microbe consortium compared to other treatments. In potato, the disease incidence percentage was significantly reduced in plants treated with the triple-microbe consortium compared to untreated control plants challenged with A. solani. Potato tubers treated with the consortium and challenged with pathogen showed significant activation of defense-related enzymes such as peroxidase (PO) at 96 h after pathogen inoculation (hapi) while, both polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) at 72 hapi, compared to the individual and dual microbial consortia-treated plants. The expression of antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT) and the accumulation of pathogenesis-related proteins such as chitinase and ß-1,3-glucanase were observed to be highest at 72 hapi in the triple microbe consortium as compared to other treatments. HPLC analysis revealed significant induction in polyphenolic compounds in triple-consortium bioprimed plants compared to the control at 72 hapi. Histochemical analysis of hydrogen peroxide (H2O2) clearly showed maximum accumulation of H2O2 in pathogen-inoculated control plants, while the lowest was observed in triple-microbe consortium at 72 hapi. The findings of this study suggest that biopriming with a microbial consortium improved plant growth and triggered defense responses against A. solani through the induction of systemic resistance via modulation of the phenylpropanoid pathway and antioxidative network.

Exploring the mechanisms of endophytic bacteria for suppressing early blight disease in tomato (Solanum lycopersicum L.).

Controlling early blight of tomatoes using endophytic bacteria is an eco-friendly and sustainable approach to manage this common fungal disease caused by Alternaria solani, Alternaria alternata, and Curvularia lunata. Endophytic bacteria are microorganisms that live inside plant tissues without causing harm and can help protect the host plant from pathogens. In this work, twenty endophytic bacterial isolates from tomato healthy plants were tested against pathogenic fungal isolates that caused early blight disease in vitro. Out of the 20 tested isolates, three (B4, B7, and B17) were considered effective isolates against the growth of fungal pathogens. The three isolates were recognized as Enterobacter cloacae HS-6 (B4), Pseudomonas gessardii HS-5 (B 7), and Pseudomonas mediterranea HS-4 (B17) using 16s-rDNA sequencing. Different concentrations of bacterial cultural diltrates at 20, 40, and 60% were tested for their antagonistic effects on the development of pathogenic fungi in vitro. The lowest dry weights of pathogenic isolates in all bacterial culture filtrates were discovered at 60%. In all culture filtrates, phenolic compounds showed the largest peak area. Under greenhouse conditions, the least disease severity of tomato early blight was found for E. cloacae and its culture filtrate compared to other treatments. Real-time PCR was used to examine the expression pattern of the defense response gene ß-1.3 glucanase gene in infected tomato plants with pathogenic fungi (control) as well as its relations with efficient biocontrol agent (E. cloacae). The expression of the gene increased substantially and significantly after three days from the inoculation-infected plants with C. lunata and E. cloacae while it reached the maximum after five days from the inoculation with A. alternata, A. solani and E. cloacae. Our study concluded that the endophytic bacterial isolate E. cloacae can be considered a promising biocontrol agent for preventing tomato early blight.

Tetraose steroidal glycoalkaloids from potato provide resistance against Alternaria solani and Colorado potato beetle.

Plants with innate disease and pest resistance can contribute to more sustainable agriculture. Natural defence compounds produced by plants have the potential to provide a general protective effect against pathogens and pests, but they are not a primary target in resistance breeding. Here, we identified a wild relative of potato, Solanum commersonii, that provides us with unique insight in the role of glycoalkaloids in plant immunity. We cloned two atypical resistance genes that provide resistance to Alternaria solani and Colorado potato beetle through the production of tetraose steroidal glycoalkaloids (SGA). Moreover, we provide in vitro evidence to show that these compounds have potential against a range of different (potato pathogenic) fungi. This research links structural variation in SGAs to resistance against potato diseases and pests. Further research on the biosynthesis of plant defence compounds in different tissues, their toxicity, and the mechanisms for detoxification, can aid the effective use of such compounds to improve sustainability of our food production.

Farmers often rely on pesticides to protect their crops from disease and pests. However, these chemicals are harmful to the environment and more sustainable strategies are needed. This is particularly true for a disease known as the early blight of potato, which is primarily treated using fungicides that stop the fungal pathogen responsible for the infection (Alternaria solani) from growing. An alternative approach is to harness the natural defence systems that plants already have in place to protect themselves. Like humans, plants have an immune system which can detect and destroy specific pathogens. On top of this, they release defence compounds that are generally toxic to pests and microbes, stopping them from infiltrating and causing an infection. In 2021, a group of researchers discovered a wild relative of the potato, known as Solanum commersonii, with strong resistance to early blight disease. Here, Wolters et al. ­ including some of the researchers involved in the 2021 study ­ set out to find how this plant defends itself from the fungus A. solani. The team found that two closely linked genes are responsible for the resistant behaviour of S. commersonii, which both encode enzymes known as glycosyltransferases. Further experiments revealed that the enzymes protect S. commersonii from early blight disease by modifying steroidal glycoalkaloids, typical defence compounds found in potato and other plants from the same family. The glycosyltransferases alter glycoalkaloids in S. commersonii by adding a sugar group to a specific part of the compound called glycone. Wolters et al. found that the glycoalkaloids from S. commersonii were able to slow the growth of other fungal pathogens that harm potatoes when tested in the laboratory. They also made plants resistant to another common destroyer of crops, the Colorado potato beetle. These findings could help farmers breed potatoes and other crops that are more resistant to early blight disease and Colorado potato beetle, as well as potentially other fungi and pests. However, further experiments are needed to investigate how these glycone-modified glycoalkaloids affect humans, and how variants of glycoalkaloids are produced and degraded in different parts of the plants. Acquiring this knowledge will help to employ these defence compounds in a safe and effective manner.

Genome and Transcriptome Analysis to Elucidate the Biocontrol Mechanism of Bacillus amyloliquefaciens XJ5 against Alternaria solani.

Early blight, caused by Alternaria solani, is an important disease affecting tomatoes. Biological control offers an environmentally friendly approach to controlling pathogens. Herein, we identified a B. amyloliquefaciens strain XJ5 and investigated its biocontrol mechanism against A. solani. A. solani growth was significantly inhibited by XJ5, with the inhibition rate of cell-free culture supernatants reaching 82.3%. Furthermore, XJ5 crude protein extracts inhibited conidia germination and altered the mycelial morphology of A. solani. To uncover the potential biocontrol mechanism of XJ5, we analyzed its genome sequence and transcriptome. The genome of XJ5 comprised a 4.16 Mb circular chromosome and two circular plasmids. A total of 13 biosynthetic gene clusters and 127 genes encoding hydrolases were identified, suggestive of the ability of XJ5 to secrete antagonistic secondary metabolites and hydrolases. Transcript analysis revealed 174 differentially expressed genes on exposing A. solani to XJ5 crude protein extracts. The expression of genes related to chitin and mannose synthesis was downregulated, indicating that XJ5 metabolites may impact chitin and mannose synthesis in A. solani. Overall, these findings enhance our understanding of the interactions between B. amyloliquefaciens and phytopathogens and pave the way for the agricultural application of this promising biocontrol agent.

Use of Trichoderma culture filtrates as a sustainable approach to mitigate early blight disease of tomato and their influence on plant biomarkers and antioxidants production.

Introduction: Alternaria solani is a challenging pathogen in the tomato crop globally. Chemical control is a rapid approach, but emerging fungicide resistance has become a severe threat. The present study investigates the use of culture filtrates (CFs) of three species of Trichoderma spp. to control this disease. Methods: Highly virulent A. solani strain and three Trichoderma fungal strains viz., T. harzianum (Accession No: MW590687), T. atroviride (Accession No: MW590689) and T. longibrachiatum (Accession No: MW590688) previously isolated by authors were used in this study. The efficacy of culture filtrates (CFs) to mitigate early blight disease were tested under greenhouse and field conditions, experiments were conducted in different seasons of 2020 using a tomato variety "doucen". Results and discussion: The CFs of T. harzianum, T. longibrachiatum, and T. atroviride significantly inhibited the in vitro mycelial growth of A. solani (62.5%, 48.73%, and 57.82%, respectively, followed by control 100%). In the GC-MS analysis of Trichoderma CF volatile compounds viz., harzianic acid (61.86%) in T. harzianum, linoleic acid (70.02%) in T. atroviride, and hydroxymethylfurfural (68.08%) in the CFs of T. longibrachiatum, were abundantly present. Foliar application of CFs in the greenhouse considerably reduced the disease severity (%) in all treatments, viz., T. harzianum (18.03%), T. longibrachiatum (31.91%), and T. atroviride (23.33%), followed by infected control (86.91%), and positively affected the plant biomarkers. In the greenhouse, the plants treated with CFs demonstrated higher flavonoids after 6 days of inoculation, whereas phenolic compounds increased after 2 days. The CF-treated plants demonstrated higher antioxidant enzymes, i.e., phenylalanine ammonia-lyase (PAL) and peroxidase (POD), after 4 days, whereas polyphenol oxidase (PPO) was higher after 6 days of inoculation, followed by healthy and infected controls. In open field conditions, disease severity in CF-treated plants was reduced in both seasons as compared to naturally infected plants, whereas CF-treated plants exhibited a higher fruit yield than controls. The present results conclude that CFs can be a potential biocontrol candidate and a promising alternative to the early blight pathogen for sustainable production.

Synergistic biocontrol of Bacillus subtilis and Pseudomonas fluorescens against early blight disease in tomato.

Early blight of tomato caused by Alternaria solani results in significant crop losses. In this study, Bacillus subtilis J3 and Pseudomonas fluorescens J8 were co-cultured as a synthetic microbial community (BCA) for synergistic biocontrol of A. solani, and the inhibition mechanism was investigated. BCA presented an inhibition ration against A. solani at 94.91%, which lowered the disease incidence by 38.26-42.87%; reduced peroxidase, catalase, superoxide dismutase activity of tomatoes by 73.11-90.22%; and promoted the biomass by 66.91-489.21%. With BCA protection, the relative expression of tomato resistance genes (including gPAL2, SWRKY, PR-10, and CHI) in roots and leaves was 12.83-90.70% lower than without protection. BCA also significantly altered the rhizosphere and phyllosphere microbial community. The abundance of potentially beneficial bacteria, including Bacillus, Pseudomonas, Arthrobacter, Lysobacter, and Rhizobium, elevated by 6.58-192.77%. They were negatively correlated with resistance gene expression, indicating their vital involvement in disease control. These results provided essential information on the synergistic biocontrol mechanism of bacteria against pathogens, which could contribute to developing novel biocontrol strategies. KEY POINTS: • Bacillus and Pseudomonas present a synergistic biocontrol effect against A. solani. • Biocontrol prevents pathogen damage and improves tomato growth and systemic resistance. • Beneficial bacteria thrive in the rhizosphere is the key to microbial regulation.

A Cas12a-based platform combined with gold nanoparticles for sensitive and visual detection of Alternaria solani.

Alternaria solani (A. solani), the causal agent of early blight in potatoes, poses a serious and persistent threat to potato production worldwide. Therefore, developing a method that can accurately detect A. solani in the early stage to avoid further spread is urgent. However, the conventional PCR-based method is not appropriate for application in the fields. Recently, the CRISPR-Cas system has been developed for nucleic acids analysis at point-of-care. Here, we propose a gold nanoparticles-based visual assay combining loop-mediated isothermal amplification with CRISPR-Cas12a to detect A. solani. After optimization, the method could detect 10-3 ng/µL genomic gene of A. solani. The specificity of the method was confirmed by discriminating A. solani from other three highly homologous pathogens. We also developed a portable device that could be used in the fields. By integrating with the smartphone readout, this platform holds significant potential in high-throughput detection of multiple pathogens in the fields.

Bacillus velezensis Strain HN-Q-8 Induced Resistance to Alternaria solani and Stimulated Growth of Potato Plant.

Bacillus velezensis HN-Q-8, isolated in our previous study, has an antagonistic effect on Alternaria solani. After being pretreated with a fermentation liquid with HN-Q-8 bacterial cell suspensions, the potato leaves inoculated with A. solani displayed smaller lesion areas and less yellowing than the controls. Interestingly, the activity levels of superoxide dismutase, peroxidase, and catalase in potato seedlings were enhanced by the addition of the fermentation liquid with bacterial cells. Additionally, the overexpression of key genes related to induced resistance in the Jasmonate/Ethylene pathway was activated by the addition of the fermentation liquid, suggesting that the HN-Q-8 strain induced resistance to potato early blight. In addition, our laboratory and field experiments showed that the HN-Q-8 strain can promote potato seedling growth and significantly increase tuber yield. The root activity and chlorophyll content of potato seedlings were significantly increased along with the levels of indole acetic acid, gibberellic acid 3, and abscisic acid upon addition of the HN-Q-8 strain. The fermentation liquid with bacterial cells was more efficient in inducing disease resistance and promoting growth than bacterial cell suspensions alone or the fermentation liquid without bacterial cells. Thus, the B. velezensis HN-Q-8 strain is an effective bacterial biocontrol agent, augmenting the options available for potato cultivation.