Identification and mechanism characterization of Streptomyces griseoaurantiacus XQ-29 with biocontrol ability against pepper southern blight caused by Sclerotium rolfsii.

Pepper southern blight, caused by Sclerotium rolfsii, is a devastating soil-borne disease resulting in significant loss to pepper, Capsicum annuum L. production. Here, we isolated an antagonistic bacterial strain XQ-29 with antifungal activity against S. rolfsii from rhizospheric soil of pepper. Combining the morphological and biochemical characteristics with the 16S rDNA sequencing, XQ-29 was identified as Streptomyces griseoaurantiacus. It exhibited an inhibition of 96.83% against S. rolfsii and displayed significant inhibitory effects on Botrytis cinerea, Phytophthora capsica and Rhizoctonia solani. Furthermore, XQ-29 significantly reduced the pepper southern blight by 100% and 70.42% during seedling and growth stages, respectively. The antifungal mechanism involved altering the mycelial morphology, disrupting cell wall and membrane integrity, accompanied by accumulation of reactive oxygen species and lipid peroxidation in S. rolfsii mycelia. Furthermore, XQ-29 promoted growth and stimulated resistance of pepper plants by increasing defense-related enzyme activities and upregulating defense-related genes. Correspondingly, XQ-29 harbors numerous functional biosynthesis gene clusters in its genome, including those for siderophores and melanin production. The metabolic constituents present in the ethyl acetate extracts, which exhibited an EC50 value of 85.48 ± 1.62 µg/mL, were identified using LC-MS. Overall, XQ-29 demonstrates significant potential as a biocontrol agent against southern blight disease.

Application of thifluzamide to stem rot in peppers: Infection and control mechanisms of sclerotium rolfsii.

In recent years, the fungal disease 'pepper stem rot', contracted from the soil-borne pathogen sclerotium rolfsii, has been increasing year by year, causing significant losses to the pepper (Capsicum annuum L.) industry. To investigate the infection mechanism of stem rot, the fungus S. rolfsii was used to infect the roots of pepper plants, and was found to affect root morphology and reduce root activity, which subsequently inhibited root growth and development. With fungal infestation, its secretions (oxalic acid, PG and PMG enzyme) were able to break normal tissues in the stem base and induced the burst of the active oxygen, which leads to injury aggravation. Morphological observations of the site of damage at the base of the stem using SEM revealed that the vascular bundles and stomata were completely blocked by hyphae, resulting in a blockade of material exchange in the plant. It was subsequently found that most of the stomata in the leaves were closed, which caused the leaves to lose their ability to photosynthesize, then turned yellow, wilt, shed, and the plant died. Commercialized fungicide thifluzamide with excellent in vitro (EC50 = 0.1 µg/mL) and in vivo curative (EC50 = 29.2 µg/mL) antifungal activity was selected to control the stem rot disease in peppers. The results demonstrated that it was able to suppress the secretion of associated pathogenic factors and reduce the outbursts of reactive oxygen species, thus reducing the damage caused by S. rolfsii at the base of the plant's stem and also enhancing the root activity of the infected plant, thereby promoting root growth. It could also inhibit fungal growth, unblock the vascular bundles and stomata, maintain a balance of material and energy exchange within the plant, and thus restore the damaged plant to its normal growth capacity. All the results will provide an adequate reference for the prevention and control of stem rot disease on peppers with thifluzamide.

Risk assessment and molecular mechanism of Sclerotium rolfsii resistance to boscalid.

Boscalid, a widely used SDHI fungicide, has been employed in plant disease control for over two decades. However, there is currently no available information regarding its antifungal activity against Sclerotium rolfsii and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 100 S. rolfsii strains collected from five different regions in China during 2018-2019 to boscalid using mycelial growth inhibition method and assessed the risk of resistance development. The EC50 values for boscalid ranged from 0.2994 µg/mL to 1.0766 µg/mL against the tested strains, with an average EC50 value of 0.7052 ± 0.1473 µg/mL. Notably, a single peak sensitivity baseline was curved, indicating the absence of any detected resistant strains. Furtherly, 10 randomly selected strains of S. rolfsii were subjected to chemical taming to evaluate its resistance risk to boscalid, resulting in the successful generation of six stable and inheritable resistant mutants. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and virulence compared to their respective parental strains. Cross-resistance tests revealed a correlation between boscalid and flutolanil, benzovindiflupyr, pydiflumetofen, fluindapyr, and thifluzamide; however, no cross-resistance was observed between boscalid and azoxystrobin. Thus, we conclude that the development risk of resistance in S. rolfsii to boscalid is low. Boscalid can be used as an alternative fungicide for controlling peanut sclerotium blight when combined with other fungicides that have different mechanisms of action. Finally, the target genes SDHB, SDHC, and SDHD in S. rolfsii were initially identified, cloned and sequenced to elucidate the mechanism of S. rolfsii resistance to boscalid. Two mutation genotypes were found in the mutants: SDHD-D111H and SDHD-H121Y. The mutants carrying SDHD-H121Y exhibited moderate resistance, while the mutants with SDHD-D111H showed low resistance. These findings contribute to our comprehensive understanding of molecular mechanisms underlying plant pathogens resistance to SDHI fungicides.

Characterizing genetic diversity of Sclerotium rolfsii isolates by biomapping of mycelial compatibility groupings and multilocus sequence analysis.

Genetic diversity in Sclerotium rolfsii is useful for understanding its population structure, identifying different mycelial compatibility groups (MCGs), and developing targeted strategies for disease management in affected crops. In our study, a comprehensive genetic analysis was conducted on 50 isolates of S. rolfsii, collected from various geographic regions and host plants. Two specific genes, TEF1α and RPB2, were utilized to assess the genetic diversity and relationships among these isolates. Notably, out of 1225 pairings examined, only 154 exhibited a compatible reaction, while the majority displayed antagonistic reactions, resulting in the formation of a barrier zone. The isolates were grouped into 10 distinct MCGs. These MCGs were further characterized using genetic sequencing. TEF1α sequences distinguished the isolates into 17 distinct clusters, and RPB2 sequences classified them into 20 clusters. Some MCGs shared identical gene sequences within each gene, while others exhibited unique sequences. Intriguingly, when both TEF1α and RPB2 sequences were combined, all 10 MCGs were effectively differentiated, even those that appeared identical with single-gene analysis. This combined approach provided a comprehensive understanding of the genetic diversity and relationships among the S. rolfsii isolates, allowing for precise discrimination between different MCGs. The results shed light on the population structure and genetic variability within this plant pathogenic fungus, providing valuable insights for disease management and control strategies. This study highlights the significance of comprehending the varied virulence characteristics within S. rolfsii isolates, categorizing them into specific virulence groups based on disease severity index (DSI) values. The association with MCGs provides additional insights into the genetic underpinnings of virulence in this pathogen. Furthermore, the identification of geographical patterns in virulence implies the influence of region-specific factors, with potential implications for disease control and crop protection strategies.Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [G. M. Sandeep] Last name [Kumar]. Author 2 Given name: [Praveen Kumar] Last name [Singh]. Also, kindly confirm the details in the metadata are correct.I confirm that the given names are accurate and presented in the correct sequence.

Deciphering the potential of Sargassum tenerrimum extract: metabolic profiling and pathway analysis of groundnut (Arachis hypogaea) in response to Sargassum extract and Sclerotium rolfsii.

Seaweed extracts have enormous potential as bio-stimulants and demonstrated increased growth and yield in different crops. The presence of physiologically active component stimulate plant stress signaling pathways, enhances growth and productivity, as well as serve as plant defense agents. The seaweed extracts can reduce the use of chemicals that harm the environment for disease management. In the present study, the Sargassum tenerrimum extract treatment was applied, alone and in combination with Sclerotium rolfsii, to Arachis hypogea, to study the differential metabolite expression. The majority of metabolites showed maximum accumulation with Sargassum extract-treated plants compared to fungus-treated plants. The different classes of metabolite compounds like sugars, carboxylic acids, polyols, showed integrated peaks in different treatments of plants. The sugars were higher in Sargassum extract and Sargassum extract + fungus treatments compared to control and fungus treatment, respectively. Interestingly, Sargassum extract + fungus treatment showed maximum accumulation of carboxylic acids. Pathway enrichment analysis showed regulation of different metabolites, highest impact with galactose metabolism pathway, identifying sucrose, myo-inositol, glycerol and fructose. The differential metabolite profiling and pathway analysis of groundnut in response to Sargassum extract and S. rolfsii help in understanding the groundnut- S. rolfsii interactions and the potential role of the Sargassum extract towards these interactions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01418-9.

Genome-wide identification and molecular characterization of CRK gene family in cucumber (Cucumis sativus L.) under cold stress and sclerotium rolfsii infection.

BACKGROUND: The plant cysteine-rich receptor-like kinases (CRKs) are a large family having multiple roles, including defense responses under both biotic and abiotic stress. However, the CRK family in cucumbers (Cucumis sativus L.) has been explored to a limited extent. In this study, a genome-wide characterization of the CRK family has been performed to investigate the structural and functional attributes of the cucumber CRKs under cold and fungal pathogen stress. RESULTS: A total of 15 C. sativus CRKs (CsCRKs) have been characterized in the cucumber genome. Chromosome mapping of the CsCRKs revealed that 15 genes are distributed in cucumber chromosomes. Additionally, the gene duplication analysis of the CsCRKs yielded information on their divergence and expansion in cucumbers. Phylogenetic analysis divided the CsCRKs into two clades along with other plant CRKs. Functional predictions of the CsCRKs suggested their role in signaling and defense response in cucumbers. The expression analysis of the CsCRKs by using transcriptome data and via qRT-PCR indicated their involvement in both biotic and abiotic stress responses. Under the cucumber neck rot pathogen, Sclerotium rolfsii infection, multiple CsCRKs exhibited induced expressions at early, late, and both stages. Finally, the protein interaction network prediction results identified some key possible interacting partners of the CsCRKs in regulating cucumber physiological processes. CONCLUSIONS: The results of this study identified and characterized the CRK gene family in cucumbers. Functional predictions and validation via expression analysis confirmed the involvement of the CsCRKs in cucumber defense response, especially against S. rolfsii. Moreover, current findings provide better insights into the cucumber CRKs and their involvement in defense responses.

Fruit rot causing by Athelia rolfsii (Sclerotium rolfsii) on jackfruit (Artocarpus heterophyllus) in China.

Artocarpus heterophyllus, known as jackfruit, was a tropical fruit and cultivated extensively as nutritional and medicinal properties in southern China in recent year. During July 2022, fruit rot was observed on the fruits at the bottom of jackfruit trees in an orchard in Zhanjiang, Guangdong (N21°9' 27" E110°17' 54") 3-4 days after typhoon. The incidence rate of fruit was about 0.3%. The initial symptom was white mycelia appearing on the surface of fruits. Mycelia with rhizomorphs spread rapidly over the fruits, formed white, often fan-shaped mats with the rapeseed size sclerotia. The infected fruits were water-soaked, quickly became rotten, and fell off. Sclerotia from disease fruits were incubated on PDA with 50 mg/L ampicillin at 25-28℃ in the dark for 2 days. Hyphae tips were transferred to get the purified isolates. Colonies with a radial growth rate of 23.2 mm/day had abundant aerial mycelia and profuse sclerotia on PDA. Hyphae of the isolates were transparent, branched, with clamp connections at septa, usually 2.9-8.3 µm (Ave. 5.8 µm) (n>30) wide. Aerial mycelia were whitish-cottony, with many narrow rhizomorphs. Spherical sclerotia developed at about 10 days after incubation, and gradually changed from white to tan-to-dark brown, and mature sclerotia were about 1.7 mm in size. The morphological characteristics was similar to those of Sclerotium rolfsii (teleomorph: Athelia rolfsii). To accurately identify the fungus, the internal transcribed spacer gene (ITS) and large subunit rRNA gene (LSU) of isolate CASS-BLM-1 were PCR amplified with primer pairs ITS1/ITS4 (White et al 1990) and V9G/LR5 (Klaubauf et al 2014). The amplicons were sequenced and deposited in GenBank with accession number OP535473 (ITS) and OP535474 (LSU). BLASTn results showed that the nucleotide sequences of ITS and LSU had high identity with corresponding sequences of A. rolfsii isolates CBS 191.62 (ITS: MH858139, 472/474(99.58%); LSU: MH869724, 882/885(99.66%)) (Vu et al 2019). Phylogenetic analysis based on ITS sequence data was obtained according to maximum likelihood method using MEGA analysis software, CASS-BLM1 was grouped in A. rolfsii clade with 100% bootstrap support value. Based on morphology and DNA sequences, the fungus was identified as A. rolfsii (anamorph: S. rolfsii). To fulfil Koch's postulates, healthy fruits on the tree and detached fruits were inoculated with 7-day-old sclerotia of isolate CASS-BLM1. Five unwound sites and five wound sites with a sterile needle were tested on each fruit and a sclerotium was put at each site. Fruits not inoculated with sclerotia were used as control the test was repeated three times. All fruit were enclosed in transparent plastic bags with sterile absorbent cotton moistened with sterile distilled water. The indoor and outdoor temperatures ranged from 25 to 30 ℃. Three days later, white mycelia were observed on all inoculation sites, and 5 days later, the inoculated fruits began to rot, while control fruits remained healthy. The same fungus with identical morphology and DNA sequences was re-isolated from the inoculated sites. Previously, A. rolfsii was reported to cause fruit rot disease on jackfruit in Bangladesh (Elahi et al 2021), this is the first report of A. rolfsii causing fruit rot on jackfruit in China. A. rolfsii is suitable for high temperature and humidity environment (Punja 1985), this report will help farmers to diagnose this disease, especially to strengthen the disease prevention during the typhoon season.

A LAMP-Based Toolbox Developed for Detecting the Major Pathogens Affecting the Production and Quality of the Chinese Medicinal Crop Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

Selection and Validation of Reference Genes for Reverse-Transcription Quantitative PCR Analysis in Sclerotium rolfsii.

Reference genes are important for the accuracy of gene expression profiles using reverse-transcription quantitative PCR (RT-qPCR). However, there are no available reference genes reported for Sclerotium rolfsii; it actually has a pretty diverse and wide host range. In this study, seven candidate reference genes (UBC, ß-TUB, 28S, 18S, PGK, EF1α and GAPDH) were validated for their expression stability in S. rolfsii under conditions of different developmental stages, populations, fungicide treatments, photoperiods and pHs. Four algorithm programs (geNorm, Normfinder, Bestkeeper and ΔCt) were used to evaluate the gene expression stability, and RefFinder was used to integrate the ranking results of four programs. Two reference genes were recommended by RefFinder for RT-qPCR normalization in S. rolfsii. The suitable reference genes were GAPDH and UBC across developmental stages, PGK and UBC across populations, GAPDH and PGK across fungicide treatments, EF1α and PGK across photoperiods, ß-TUB and EF1α across pHs and PGK and GAPDH across all samples. Four target genes (atrB, PacC, WC1 and CAT) were selected for the validation of the suitability of selected reference genes. However, using one or two reference genes in combination to normalize the expression of target genes showed no significant difference in S. rolfsii. In short, this study provided reliable reference genes for studying the expression and function of genes in S. rolfsii.

Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot.

BACKGROUND: Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. METHODS: We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. RESULTS: P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. CONCLUSIONS: P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

Baseline sensitivity and bioactivity of tetramycin against Sclerotium rolfsii isolates in Huanghuai peanut-growing region of China.

Peanut stem rot caused by Sclerotium rolfsii is a serious soil-borne disease and poses a threat to the peanut production. The antibiotic fungicide tetramycin has a broad antifungal spectrum against multiple pathogens and possess low environmental risks. In current study, a total of 250 isolates collected from Huanghuai peanut-growing region of China (Henan, Shandong and Hebei Province) were used to establish the baseline sensitivity of S. rolfsii to tetramycin. The baseline sensitivity curve was unimodal and distributed from 0.01 to 0.36 mg/L, with a mean EC50 (50% effective concentration) value of 0.11 ± 0.06 mg/L. Tetramycin also had strong inhibitory activity on the formation and germination of sclerotia. There was no significant correlation of S. rolfsii sensitivity to tetramycin and other commonly used SDHI (succinate dehydrogenase inhibitor), QoI (quinone outside respiration inhibitor) and DMI (demethylation inhibitor) fungicides. Moreover, tetramycin significantly increased the cell membrane permeability and reduced the oxalate acid content. Greenhouse experiments showed that tetramycin has both protective and curative efficacy against S. rolfsii, while protective efficacy was higher than curative efficacy. Anyhow, the bioactivity of tetramycin is similar (curative efficacy) or higher (protective efficacy) than the control fungicide validamycin. In terms of application method, root drench may be more suitable for tetramycin than spraying, because root drench of tetramycin obtained a higher efficacy. These results indicated that tetramycin may be a potential alternative fungicide for the efficient control of peanut stem rot.

Piperine, Reserpine and ß-Sitosterol Attenuate Stem Rot (Sclerotium rolfsii Sacc.) of Groundnut by Inducing the Secretion of defense Enzymes and Phenolic Acids.

Groundnut stem rot caused by Sclerotium rolfsii is a major constraint as it affects the productivity. Although managing this disease using synthetic fungicides is a more feasible method, environmental pollution and side effects caused by them demand some safe fungicides. Seven phytochemicals piperine, quercetin, reserpine, atropine sulfate, ß-sitosterol, ethyl protocatechuate and salicylic acid were initially tested against S. rolfsii under in vitro methods. All the compounds exhibited significant effects on mycelial inhibition (except atropine sulfate), sclerotial development, ooze formation, maturity, sclerotial number and germination of S. rolfsii. The more active compounds, piperine, reserpine and ß-sitosterol were then evaluated under glasshouse condition by adopting various application methods (seed treatment, foliar application and micro-injection at 2000 µg/mL) on groundnut plants challenged with and without S. rolfsii. All the treatments effectively reduced the plant mortality when tested every 15 days of infection with S. rolfsii. However, the magnitude of reduction varied among the treatments, with the mortality ranging between 9.37 % and 29.68 % compared to the control (40.68 %). The piperine-microinjected plants recorded minimum mortality (3.12 %). The defense enzymes (PAL and PPO) and key end products such as phenolics (total and individual) were determined in the leaf samples collected after 24, 48 and 72 h of infection with S. rolfsii to understand the systemic resistance induction effect. An increase in PAL and PPO activity was observed in all the samples. While microinjection of ß-sitosterol caused a maximum PAL induction, piperine caused a maximum PPO activity. Further, samples of piperine treated group showed higher induction of phenolic acids (86.46 µg g-1 micro-injection) compared to ß-sitosterol and reserpine groups in all the methods. When the samples were analysed (HPLC) for individual phenolic acids, maximum accumulation of various acids was observed in the samples collected after 48 h. Tannic and gallic acids were found to be accumulated in higher quantities in most of the samples. The maximum accumulation of phenolic acids was found in micro-injected samples. These results verified the reduction of mortality through the induction of defensive chemicals by the action of phytochemicals. Thus, the study recommends the use of these natural molecules for the integrated management of stem rot of groundnut after necessary field trials.

First Report of Sclerotium rolfsii Causing Southern Blight on Stephania tetrandra in China.

Stephania tetrandra S. Moore is a perennial liana and is widely cultivated in southern China for traditional Chinese medicine as a diuretic, anti-inflammatory, and antirheumatic treatment (Jiang et al. 2020). In August 2021, it was observed that a severe stem rot disease affected St. tetrandra cultivated in Anfu, Jiangxi province, China (114°27'26" E, 27°22'46" N). The disease symptoms included constriction and rot at the base of the stem, and covered with a layer of white mycelia. The plants above-ground finally wilted and dried with a disease incidence ranging from 8% to 16%. Lots of dried plants formed withered patches of field. Sections (1.0~2.0 cm) from browning stem tissues were surface-disinfected with 75% ethanol for 15 s, followed by 60 s in 4% NaClO, rinsed twice in sterile water, dried on sterilized filter paper, placed on potato dextrose agar (PDA), and incubated at 26°C in the dark for 3 days. A white rhizomorphic fungal mycelium, that is similar to the mycelium of strain FJSR0 on the surface of an infected plant in the field, was isolated from the cultured tissues with 67% frequency. When incubated on PDA, white and fluffy mycelia with even margins and a slight halo formed. Mycelia-produced clamp connections were observed. Colonies grew quickly and covered the dish (diameter: 9 cm) in 5 or 6 days. After that, sclerotia were initially white, then turned yellow, and chestnut brown at maturity. Spherical and subspherical sclerotia were observed after 8 days, with each plate containing 448 to 634 sclerotia (0.8 to 1.4 mm diameter; mean = 0.94 mm; n = 50). On the basis of morphology, the pathogen was similar to Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough] (Sun et al. 2020; Ling et al. 2021). For molecular confirmation, the internal transcribed spacer (ITS) region with approximately 680 bp was amplified from strains FJRS0 and FJRS1 using primers ITS1/ITS4 (White et al. 1990). Two distinct types (different in one SNP and one 1-bp InDel) of ITS sequences were obtained from each isolate, and all isolates contain the two types (FJSR0: ON972516, ON972517; FJSR1: ON972520, ON972518). BLAST analysis of each type found that the hits, with identities >99%, are A. rolfsii except for two Sc. delphinii sequences (GU567775.1 and MK073010.1). Phylogenetic analysis placed strains FJSR0 and FJSR1 in the same clade as Sc. rolfsii but away from Sc. delphinii based on the previous method (Sun et al. 2021). Both morphological and molecular characteristics confirmed that the strains were Sc. rolfsii. For pathogenicity tests, PDA plugs (8 mm in diameter) covered with 5-day-old fungal mycelium were inoculated at the stem bases of three healthy St. tetrandra seedings and incubated at 26℃ and relative humidity of 80%. On the fifth day, inoculated plants were wilting. The infected stem bases turned brown to black and constricted as previously observed in the field. Some leaves, infected by the mycelium expanded from the PDA plugs, developed an orange and irregular spot. Sclerotia were observed 20 days post inoculation. In contrast, the leaves and stems of non-inoculated control plants remained symptomless. Pathogenicity tests were repeated three times. The fungus was reisolated consistently from each symptomatic tissue, thus completing Koch's postulates. Although Sc. rolfsii has been previously reported to cause a southern blight symptoms on vegetables, ornamentals, grass, and medicinal and leguminous crops (Sun et al. 2020; Ling et al. 2021), this is the first report of Sc. rolfsii causing similar symptoms of southern blight on St. tetrandra in China.

Identification and Characterization of Sclerotium delphinii Causing Southern Blight on Aconitum kusnezoffii in Northeast China.

Aconitum kusnezoffii is a perennial medicinal plant that belongs to the Ranunculaceae family and is distributed mainly in Northeast and North China. In July 2018, a typical southern blight disease of A. kusnezoffii was observed in commercial fields of Qingyuan County, Fushun City, Liaoning Province, China. The fungus mainly infected stem base and tuberous roots of the plant by wrapping the hyphae and absorbing nutrition, resulting in tuberous root wilted or whole plant death. Morphological characteristics of colony and sclerotia of three representative strains isolated from the diseased plants differed from those of Sclerotium rolfsii isolated from A. carmichaelii. Sclerotia were large (0.8 to 5.1 mm), reddish-brown, and irregular and had pitted surfaces, and the hyphae were white, compact, or fluffy, with a growth rate ranging from 8.0 to 10.1 mm/day. Phylogenetic analysis of the internal transcribed spacer and the large subunit sequences of Akln6, Akln9, and Akln15 showed that three strains isolated from A. kusnezoffii formed a unique and well-supported clade that groups with the reference isolates of S. delphinii. Based on phylogenetic analysis and cultural and morphological characteristics, the three isolates of A. kusnezoffii were identified as S. delphinii. The optimum temperature for mycelial growth of the three tested isolates was 30°C, and sclerotia formed and matured more easily at 20°C. Light promoted the growth of mycelial, whereas dark was beneficial to the formation and maturation of sclerotia. The pathogenicity of S. delphinii showed stronger than S. rolfsii at low temperature (20°C). This is the first report of S. delphinii causing southern blight on A. kusnezoffii in China, and this finding provides a basis for disease-accurate diagnosis and the development of effective management strategies.

Comparison of Current Peanut Fungicides Against Athelia rolfsii Through a Laboratory Bioassay of Detached Plant Tissues.

Southern stem rot of peanut, caused by Athelia rolfsii, is an important fungal disease that impacts peanut production worldwide. Foliar-applied fungicides are used to manage the disease, and several fungicides have been recently registered for southern stem rot control in peanuts. This study compared fungicidal, residual, and potential systemic activity of current fungicides against A. rolfsii using a laboratory bioassay. Peanut plants grown in the field were treated with eight fungicides approximately 90 days after planting, and plants were collected for the laboratory bioassay weekly for 5 weeks following application. Peanut plants were separated into the newest fully mature leaf present at sample collection, the second newest fully mature leaf present at the time of fungicide application, the upper stem, and the crown tissues. Each plant tissue was inoculated with A. rolfsii then incubated at 30°C for 2 days. Lesion length was measured, and percent inhibition of fungal growth by each fungicide relative to the control was calculated. All fungicides provided the greatest inhibition of A. rolfsii on leaf tissues that were present at the time of fungicide application, followed by the newly grown leaf and upper stem. Little inhibition occurred on the crown. Fungal inhibition decreased at similar rates over time for all fungicides tested. Succinate dehydrogenase inhibitors provided less basipetal protection of upper stems than quinone outside inhibitor or demethylation inhibitor fungicides. Properties of the fungicides characterized in this study, including several newly registered products, are useful for developing fungicide application recommendations to maximize their efficacy in controlling both foliar and soilborne peanut diseases.

Genome sequencing and comparative genomic analysis of highly and weakly aggressive strains of Sclerotium rolfsii, the causal agent of peanut stem rot.

BACKGROUND: Stem rot caused by Sclerotium rolfsii is a very important soil-borne disease of peanut. S. rolfsii is a necrotrophic plant pathogenic fungus with an extensive host range and worldwide distribution. It can infect peanut stems, roots, pegs and pods, leading to varied yield losses. S. rolfsii strains GP3 and ZY collected from peanut in different provinces of China exhibited a significant difference in aggressiveness on peanut plants by artificial inoculation test. In this study, de-novo genome sequencing of these two distinct strains was performed aiming to reveal the genomic basis of difference in aggressiveness. RESULTS: Scleotium rolfsii strains GP3 and ZY, with weak and high aggressiveness on peanut plants, exhibited similar growth rate and oxalic acid production in laboratory. The genomes of S. rolfsii strains GP3 and ZY were sequenced by Pacbio long read technology and exhibited 70.51 Mb and 70.61 Mb, with contigs of 27 and 23, and encoded 17,097 and 16,743 gene models, respectively. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires, which might be associated with aggressiveness, differed between GP3 and ZY. There were 58 and 45 unique pathogen-host interaction (PHI) genes in GP3 and ZY, respectively. The ZY strain had more carbohydrate-active enzymes (CAZymes) in its secretome than GP3, especially in the glycoside hydrolase family (GH), the carbohydrate esterase family (CBM), and the polysaccharide lyase family (PL). GP3 and ZY also had different effector candidates and putative secondary metabolite synthetic gene clusters. These results indicated that differences in PHI, secreted CAZymes, effectors and secondary metabolites may play important roles in aggressive difference between these two strains. CONCLUSIONS: The data provided a further understanding of the S. rolfsii genome. Genomic comparison provided clues to the difference in aggressiveness of S. rolfsii strains.

Gliotoxin Is an Important Secondary Metabolite Involved in Suppression of Sclerotium rolfsii of Trichoderma virens T23.

Sclerotium rolfsii causes destructive soilborne disease in numerous plant species, and biological control may be a promising and sustainable approach for suppressing this widespread pathogen. In this study, the antagonistic effect against S. rolfsii of 10 Trichoderma strains was tested by the dual culture method, and a gliotoxin-producing strain, T. virens T23, was shown to be the most effective, inhibiting growth of S. rolfsii in vitro by 70.2%. To clarify the antagonistic mechanism and gliotoxin biosynthesis regulation of T23, a gliotoxin-deficient mutant was constructed via Agrobacterium tumefaciens-mediated gene knockout in vivo. As expected, disruption of the gene located in the putative gliotoxin biosynthesis gene cluster, gliI-T, resulted in gliotoxin deficiency and attenuation of the antagonistic effect against S. rolfsii, indicating that gliotoxin biosynthesis is regulated by gliI-T and that gliotoxin is an important antifungal metabolite of T23. Transmission electron microscopy revealed that gliotoxin treatment caused marked alterations of the hyphal cells of S. rolfsii depending on the drug concentration, whereby one of the prominent structural alterations was a reduction in the number and length of mitochondrial cristae. When S. rolfsii was exposed to 30 µg/ml of gliotoxin for 12 h, striking plasmolysis and ultrastructural changes were induced. The results demonstrated that gliotoxin is an important secondary metabolite of T. virens T23 in its antagonism against S. rolfsii.

Baseline Sensitivity and Control Efficacy of Strobilurin Fungicide Pyraclostrobin Against Sclerotium rolfsii.

Sclerotium rolfsii is a fungi pathogen of southern blight with broad host range. The quinone outside inhibitor fungicide pyraclostrobin was officially approved for controlling many diseases in 2015. In this study, baseline sensitivity of S. rolfsii to pyraclostrobin was established by measuring the 50% effective concentration (EC50) values of 155 isolates of S. rolfsii. The EC50 values ranged from 0.0291 to 1.0871 µg/ml, with a mean EC50 of 0.4469 ± 0.2490 µg/ml (mean ± standard deviation). In a preventive fungicide in vitro experiment and a field experiment, pyraclostrobin preventive efficacy reached 90% and 80%, respectively, which were much higher than that of the control agent carbendazim. Curative efficacy of pyraclostrobin was significantly lower than its preventive efficacy. Pyraclostrobin at 0.1, 0.5, and 2 µg/ml significantly reduced the number of sclerotia produced on potato dextrose agar medium, but had no significant influence on their total weight. Pyraclostrobin had no significant influence on mycelial cell membrane permeability, but it significantly reduced oxalate secretion and protein synthesis of S. rolfsii. Our findings are of great significance for resistance monitoring of S. rolfsii and also provide new insight into the action mechanism of pyraclostrobin against S. rolfsii.

Southern Blight on Medicago sativa Caused by Sclerotium rolfsii in North China.

Alfalfa (Medicago sativa L.) is one of the most important perennial leguminous forages in many countries, known by its high feed value and yield potential. With the increasing demand for feed, alfalfa has been planted all over China. However, an increasingly serious alfalfa disease was observed and may restrict the development of the alfalfa industry in North China. In August 2019, an emerging alfalfa disease with symptoms resembling southern blight was observed in Jiaozhou experimental base (Jiaozhou Modern Agricultural Science and Technology Demonstration Park) of Qingdao Agricultural University (Qindao, Shandong province, China). The infected plants showed dark brown lesions on the stems and yellowing and wilting of the leaves. The pathogen produced white fluffy mycelia, and later sclerotia on stems and roots; the disease affected up to 25% of the plants and causes bare spots filled with weeds (Figure S1). Typical symptomatic tissues were brought back to the laboratory for pathogen isolation and identification. Fragments (3-5mm2) of root tissues were excised from lesions on the symptomatic roots and their surfaces were disinfested by sequential dipping in 70% ethanol for 30 s and in 2% NaClO for 3 min, then the fragments were rinsed in sterile water five times and cultured on potato dextrose (PDA) medium amended with streptomycin sulfate (0.1mg/mL). Cultures were incubated at 28°C in the dark and purified in PDA medium for three times. A representative strain (coded as CZL1) was isolated from the root rot of the diseased plant. After four days incubation on PDA, CZL1 formed white fluffy aerial mycelium 5.6-6 cm in diameter typical of S. rolfsii. After 15 to 20 days, abundant round sclerotia approximately1-3 mm in diameter were produced on the surface of the culture (Figure S2). The sclerotia were white at first and then gradually turned dark brown. To confirm the identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA region of the fungus was amplified using the primers ITS1/ITS4 (White et al.1990), and the elongation factor-1a gene (EF1a) was amplified using primers EF1-983F/EF1-2218R (Rehner and Buckley 2005). Then the PCR amplicons were cloned into the pCE2 TA/Blunt-Zero vector. The isolate was determined to contain two distinct sequence types for each gene. The results of ITS (MT812692, MT812693) and EF1a (MT846496 and MT846497) sequences were deposited in GenBank. DNA analysis revealed that the two ITS sequences were more than 99% identical to Athelia rolfsii (MN872304) in the NCBI GenBank database, and two EF1a sequences were 99% identical to the A. rolfsii EF1a sequence MN702789 and KP982854. To fulfill Koch's postulates, infected sorghum grain was placed near the roots of 15 40-day-old healthy alfalfa seedlings split into 3 pots with the same number of seedlings receiving a control treatment of sterilized sorghum grain. All plants were incubated in growth chamber at 24±1°C with 14-h-photoperiod (85% relative humidity). After 10-15 days, blight symptoms identical to those in the field were observed on inoculated plants, whereas those control plants were symptomless (Figure S2). S. rolfsii was successfully re-isolated from the inoculated plants and molecularly characterized as described above. Based on disease symptoms, fungal colonies, the ITS and EF1a sequence, and pathogenicity to the host, this fungus was identified as S. rolfsii (teleomorph Athelia rolfsii). To our knowledge, this is the first report of S. rolfsii as the causal agent of southern blight of alfalfa in North China, and it is also the first report of southern blight on alfalfa caused by S. rolfsii in China since 1996 observed in Guizhou province (Mo and Luo 1996).

Peanut disease epidemiology under dynamic microclimate conditions and management practices in North Florida.

Diverse field characteristics, weather patterns, and management practices can result in variable microclimates. The objective was to relate in-field microclimate conditions with peanut diseases and yield and determine the effect of irrigation and fungicides within these environments. Irrigation did not have a major impact on disease and yield. Stem rot (Athelia rolfsii) and early (Passalora arachidicola) and late (Nothopassalora personata) leaf spot were most affected by changes in environmental patterns across seasons. Average non-treated stem rot was 12.9% in 2017 which dropped considerably in 2018 to 0.2% but emerged again in 2019 to 3.2%. Stem rot incidence varied across the field, and the response to fungicides depended on management zone. Leaf spot defoliation in non-treated plots was severe in 2019 reaching an average of 73% at 126 days after planting but only reached 15% in 2017 and 35% in 2019 at the same stage. A low-input fungicide schedule was able to reduce foliar disease in all zones and seasons, but the microclimatic conditions in the low-lying area favored leaf spot in 2017 and 2018 although not in the dryer 2019 season. Seasonal differences in disease and plant growth affected the level of protection against average yield loss using a standard low-input program which in 2017 (527 kg/ha) was not as great as 2018 (2,235 kg/ha) or 2019 (1,763 kg/ha). Disease prediction models built on dynamic environmental factors in the context of multiple pathogens and natural field conditions could be developed to improve within-season management decisions for more efficient fungicide inputs.