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Biological control agent Bacillus subtilis formulated as Theia is registered for control of fungal and bacterial diseases of fruit crops. Combinations of Theia and strategic concentrations of two demethylation inhibitor (DMI) fungicides were investigated to explore potential synergisms. Bacteria were cultured in nutrient broth and combined with technical grades and two formulations of propiconazole (emulsifiable concentrate [EC] and wettable powder) and metconazole (EC and water-dispersible granule) at 0, IO, 50, 100, and 150 ug/ml of active ingredient. After cocultivation, the optical density (OD«x)) and colony forming units (CFU/ml) were evaluated. In contrast to EC formulations, the wettable powder or water-dispersible granule formulations at 10 or 50 pg/ml of both DMIs did not affect vegetative cell growth. The mixture of Theia and each formulated DMI at 50 ug/ml of active ingredient resulted in a significant reduction of Moniliniafructicola lesion development on apple, Colletotrichum siamense lesion development on cherry, and Botrytis cinerea lesion development on cherry. The combination of Theia with EC formulations showed weaker disease reduction due to antagonism. Only Theia plus non-EC formulated propiconazole and metconazole significantly reduced brown rot disease incidence of apple compared with the respective solo treatments and anthracnose disease incidence of cherry compared with the untreated control. Our results indicated that at least some DMI fungicides possess bactericidal effects depending on the formulation and concentration. The combination of Theia with a lower-than-label-rateconcentration (50 ug/ml) of the DMI fungicides propiconazole and metconazole showed potential for synergistic effects, especially when non-EC formulations were used. [ABSTRACT FROM AUTHOR]

Armillaria root rot (ARR) poses a significant threat to the long-term productivity of stone-fruit and nut crops in the predominant production area of the United States. To mitigate this issue, the development of ARR-resistant and horticulturally-acceptable rootstocks is a crucial step towards the maintenance of production sustainability. To date, genetic resistance to ARR has been found in exotic plum germplasm and a peach/plum hybrid rootstock, 'MP-29'. However, the widely-used peach rootstock Guardian® is susceptible to the pathogen. To understand the molecular defense mechanisms involved in ARR resistance in Prunus rootstocks, transcriptomic analyses of one susceptible and two resistant Prunus spp. were performed using two causal agents of ARR, including Armillaria mellea and Desarmillaria tabescens. The results of in vitro co-culture experiments revealed that the two resistant genotypes showed different temporal response dynamics and fungus-specific responses, as seen in the genetic response. Gene expression analysis over time indicated an enrichment of defense-related ontologies, including glucosyltransferase activity, monooxygenase activity, glutathione transferase activity, and peroxidase activity. Differential gene expression and co-expression network analysis highlighted key hub genes involved in the sensing and enzymatic degradation of chitin, GSTs, oxidoreductases, transcription factors, and biochemical pathways likely involved in Armillaria resistance. These data provide valuable resources for the improvement of ARR resistance in Prunus rootstocks through breeding. [ABSTRACT FROM AUTHOR]

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories, i.e., low resistance (LR, 6.5 ≤ EC50 < 13.0 μg/mL), moderate resistance 1 (MR-1, 13.0 ≤ EC50 < 25.0 μg/mL), and moderate resistance 2 (MR-2, 25.0 ≤ EC50 < 35.0 μg/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the VelB-VeA-LaeA complex played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT. Author summary: Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Here, we explored the mechanisms of low IPT resistance in M. oryzae. Combining the whole genome sequencing and genetic transformation, we identified a conserved MoVelB gene, encoding the velvet family transcription factor to be a determinant for IPT toxicity. We further demonstrated that the deformation of the VelB-VeA-LaeA complex conferred the low IPT-resistance in M. oryzae, most likely through down-regulating the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT. This study improved our understanding of the resistance mechanism as well as the mode of action of IPT which will be helpful for making suitable strategies to manage the emerging resistance of IPT in M. oryzae. [ABSTRACT FROM AUTHOR]

Cut roses are highly valuable ornamentals and their profitability depends on the flower postharvest performance. Bent neck symptoms in roses make them unmarketable and are typically related to physiological disorders, bacteria accumulation in the vase solution, and Botrytis cinerea infection. Unusual bent neck symptoms were observed in 4.7% of 'Orange Crush' roses from two commercial shipments, resulting in complete flower collapse. This research was aimed to determine the causal agent of the bent neck symptoms. Following incubation in a humid chamber, symptomatic roses evolved in water-soaked lesions with the presence of white mycelium and sclerotia development. Fungal isolations and molecular characterization were performed and Sclerotinia sclerotiorum was identified as the causal agent of rose bent neck. Therefore, when bent neck symptoms are observed, S. sclerotiorum incidence should be considered to avoid possible outbreaks. [ABSTRACT FROM AUTHOR]

Emerging fungal pathogens have always been an issue of concern in southeastern U.S. strawberry production. In 2023, an unusual outbreak of Gnomonia leaf blotch occurred at one North Carolina (NC) and multiple South Carolina (SC) strawberry farms and marked the first report of its occurrence in SC. Molecular identification and phylogenetic analysis of isolates from multiple locations identified the fungus Gnomoniopsis fructicola as the causal agent. In vitro germination of G. fructicola progressed slowly and remained less than 40% even after 24 h of incubation. Similarly, germ tube growth was slow compared to other pathogens. Slow symptom development on strawberry leaves of young strawberry plants grown in the greenhouse started 5 weeks after inoculation. Once the pathogen established on greenhouse plants, leaf necrosis forming blotches was observed. The baseline sensitivity of G. fructicola isolates to commonly used chemical classes of fungicides was assessed. Propiconazole, cyprodinil, pyraclostrobin, and fludioxonil were highly effective in mycelial growth assays with EC50 values < 0.01 µg/ml. Iprodione and thiophanate-methyl were also effective with EC50 values ranging from 0.05 to 1.38 and 2.01 to 23.96 µg/ml, respectively. Fluopyram and fenhexamid were ineffective with EC50 values >100 µg/ml. Based on conversations with the producers, the disease outbreak was linked to transplants from the same nursery source. This study reports for the first time the presence of Gnomonia leaf blotch in South Carolina and provides valuable insights into chemical management options for G. fructicola.

Background: Peach brown rot caused by Monilinia fructicola severely affects the quality and yield of peach, resulting in large economic losses worldwide. Methyl benzimidazole carbamate (MBC) fungicides and sterol demethylation inhibitor (DMI) fungicides are among the most applied chemical classes used to control the disease but resistance in the target pathogen has made them risky choices. Timely monitoring of resistance to these fungicides in orchards could prevent control failure in practice., Results: In the current study, we developed methods based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems to detect MBC and DMI resistance based on the E198A mutation in the β-tubulin (MfTub2) gene and the presence of the Mona element in the upstream region of the MfCYP51, respectively. For MBC resistance, RPA primers were designed that artificially incorporated PAM sites to facilitate the CRISPR/Cas12a reaction. Subsequently, specific tcrRNAs were designed based on the E198A mutation site. For the detection of the Mona element, we designed RPA primers M-DMI-F2/M-DMI-R1 that in combination with crRNA1 detected 'Mona' and distinguished resistant from sensitive strains., Conclusion: Both methods exhibited high sensitivity and specificity, requiring only a simple isothermal device to obtain results within 1 h at 37 °C. The FQ-reporter enabled visualization with a handheld UV or white light flashlight. This method was successfully used with purified DNA from lab cultures and crude DNA from symptomatic fruit tissue, highlighting its potential for on-site detection of resistant strains in orchards. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Brown rot caused by Monilinia fructicola is one of the most important diseases affecting peach production in the southeastern USA. Management often involves the use of demethylation inhibitor (DMI) fungicides, but efficacy can be compromised due to overexpression of the MfCYP51 gene encoding the 14α-demethylase of the ergosterol biosynthesis pathway. This study aimed to investigate the influence of the biorational fungicide Howler EVO containing Pseudomonas chlororaphis ASF009 metabolites, on the expression of MfCYP51 in M. fructicola and associated synergy with a DMI fungicide for control of DMI-resistant strains. Mycelia from two DMI-sensitive and three DMI-resistant M. fructicola isolates were exposed or not to propiconazole (0.3 µg/ml), Howler (78.5 µg/ml), or the combination propiconazole + Howler for 6 h prior to RNA extraction. Real-time PCR indicated that Howler reduced the constitutive expression of MfCYP51 in DMI sensitive and two of three DMI-resistant isolates. Propiconazole-induced expression of the DMI target gene was significantly reduced by Howler and by the mixture of Howler plus propiconazole in all isolates. Detached fruit studies on apple revealed that the combination of Howler plus a reduced label rate of Mentor (50 µg/ml propiconazole) was synergistic against brown rot caused by a DMI-resistant isolate in high and low inoculum spore concentration experiments (synergy values of 40.1 and 4.9, respectively). We hypothesize that the synergistic effects against M. fructicola resistant to DMI fungicides based on MfCYP51 gene overexpression can be attributed to reduced 14α demethylase production due to transcription inhibition, which may necessitate fewer DMI fungicide molecules to arrest fungal growth. The use of Howler /DMI mixtures for brown rot control warrants further investigation because such mixtures could potentially allow for reduced DMI fungicide use rates in the field without compromising yield or increased resistance selection.

Bacterial spot is one of the most serious diseases of peach caused by the pathogen Xanthomonas arboricola pv. pruni (XAP), leading to early defoliation and unmarketable fruit. The pathogen can overwinter in peach twigs and form spring cankers, which are considered the primary inoculum source for early season leaf and fruitlet infection. The amount of overwintering bacterial inoculum plays a critical role for the bacterial spot development, but no reliable quantification method is available. Thus, we developed a long-amplicon propidium monoazide (PMA)-quantitative PCR (qPCR) assay for specific detection of viable XAP cells. The optimized PMA-qPCR assay used 20 μM of PMAxx for pure bacterial suspensions and 100 μM for peach twig tissues. The Qiagen Plant Pro Kit with an additional lysozyme digestion step was the DNA extraction protocol that yielded the best detection sensitivity with the bacteria-spiked peach twig extracts. The PMA-qPCR assay was tested with different mixtures of viable and heat-killed XAP cells in pure bacterial suspensions and bacteria-spiked peach twig tissues. The results showed that this assay enabled sensitive, specific, and accurate quantification of viable XAP cells as low as 10 3 CFU/ml with the presence of up to 10 7 CFU/ml of dead XAP cells, while suppressing the amplification of DNA from dead cells. For mixtures of viable and dead cells, the PMA-qPCR results were linearly correlated with the predicted concentrations of viable XAP ( R 2 > 0.98). Thus, the PMA-qPCR assay will be a suitable tool for quantifying overwintering XAP population on peach trees., Competing Interests: The author(s) declare no conflict of interest.

Sterol demethylation inhibitor (DMI) fungicides continue to be essential components for the control of brown rot of peach caused by Monilinia fructicola in the United States and worldwide. In the southeastern United States, resistance to DMIs had been associated with overexpression of the cytochrome P450 14α-demethylase gene Mf CYP51 as well as the genetic element Mona, a 65 bp in length nucleotide sequence located upstream of Mf CYP51 in resistant isolates. About 20 years after the first survey, we reevaluated sensitivity of M. fructicola from South Carolina and Georgia to propiconazole and also evaluated isolates from Alabama for the first time. A total of 238 M. fructicola isolates were collected from various commercial and two experimental orchards, and sensitivity to propiconazole was determined based on a discriminatory dose of 0.3 μg/ml. Results indicated 16.2, 89.2, and 72.4% of isolates from Alabama, Georgia, and South Carolina, respectively, were resistant to propiconazole. The detection of resistance in Alabama is the first report for the state. All resistant isolates contained Mona, but it was absent from most sensitive isolates. It was unclear if the resistance frequency had increased in South Carolina and Georgia. However, the resistance levels (as assessed by the isolate frequency in discriminatory dose-based relative growth categories) did not change notably, and no evidence of other resistance genotypes was found. Analysis of the upstream Mf CYP51 gene region in the resistant isolate CF010 revealed an insertion sequence described for the first time in this report. Our study suggests that current fungicide spray programs have been effective against increasing resistance levels in populations of M. fructicola and suppressing development of new resistant genotypes of the pathogen., Competing Interests: The author(s) declare no conflict of interest.

A new Neopestalotiopsis sp. was recently reported causing outbreaks of leaf spot and fruit rot on strawberry in Florida, Georgia, and South Carolina. In contrast to other Pestalotiopsis pathogens, the new species appears more aggressive and destructive on strawberry. Current chemical options for management are disease suppressive at best, and affected growers have been experiencing major yield losses. In this study, we developed a molecular method based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) for identification of the new Neopestalotiopsis sp. from strawberry. Isolates of the new Neopestalotiopsis sp. collected in Florida; isolates of N. rosae , N. honoluluana , N. ellipsopora , N. saprophytica , N. samarangensis , and P. rhododendri ; and isolates from South Carolina suspected to be the new Neopestalotiopsis sp. were included in this study. This method is based on PCR amplification of a β-tubulin gene fragment using a previously published set of primers (Bt2a and Bt2b), followed by use of the restriction enzyme BsaWI. The enzyme cuts the PCR product from the new Neopestalotiopsis sp. twice, yielding fragments of 290 base pairs (bp) and 130 and 20 bp in size, whereas fragments from other species are only cut once, yielding fragments of 420 and 20 bp. This method will aid research labs and diagnostic clinics in the accurate and fast identification of the aggressive Neopestalotiopsis sp. variant from strawberry., Competing Interests: The author(s) declare no conflict of interest.

Glomerella leaf spot (GLS) and bitter rot (BR), caused by Colletotrichum spp., are major diseases on apple in southern Brazil. Among integrated pest management tools for disease management in commercial orchards, fungicides remain an important component. This study aimed to identify Colletotrichum spp. from cultivar Eva in Paraná state orchards; evaluate their in vitro sensitivity to cyprodinil, tebuconazole, iprodione, and fluazinam; and determine the baseline in vitro sensitivity of these isolates to benzovindiflupyr and natamycin. Most isolates belonged to Colletotrichum melonis and C. nymphaeae of the C. acutatum species complex. The two species varied in sensitivity to fluazinam and tebuconazole, but no variability was found for any other fungicide. The lowest 50% effective concentration (EC 50 ) values of Colletotrichum spp. were observed for cyprodinil (mean EC 50 < 0.02) and benzovindiflupyr (mean EC 50 < 0.05); EC 50 values were intermediate for fluazinam (mean EC 50 < 0.33) and tebuconazole (mean EC 50 < 0.14), and they were highest for natamycin (mean EC 50 < 5.56) and iprodione (mean EC 50 > 12). Cyprodinil and fluazinam are registered for use in Brazil for apple disease management but not specifically for GLS and BR. Tebuconazole is one of the few products registered for Colletotrichum spp. control in apples. In conclusion, flowers and fruitlets can serve as sources of inoculum for GLS and BR disease; C. acutatum was the predominant species complex in these tissues; cyprodinil and fluazinam applications may suppress GLS and BR; and benzovindiflupyr and natamycin warrant further investigation for GLS and BR disease control of apple due to comparably high in vitro sensitivity.

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni , causes yield loss every year in southeastern U.S. peach orchards. Management is mainly driven by season-long applications of copper-based products, site location, and choice of cultivar. Although tolerance to copper has not been reported in X . arboricola pv. pruni in the United States, adaptation of populations from frequent use is a concern. We collected X . arboricola pv. pruni from shoot cankers, leaves, and fruit of cultivar O'Henry over 2 years from three conventional farms and one organic farm in South Carolina, one orchard per farm. The four farms had been using copper extensively for years to control bacterial spot. X . arboricola pv. pruni was isolated from four canker types (bud canker, tip canker, nonconcentric canker, and concentric canker) in early spring (bud break), as well as from leaf and fruit tissues later in the season at the phenological stages of pit hardening and final swell. X . arboricola pv. pruni was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. X . arboricola pv. pruni isolates were assessed for sensitivity to copper using minimal glucose yeast agar and nutrient agar amended with 38 μg/ml or 51 μg/ml of Cu 2+ . Two phenotypes of copper tolerance in X . arboricola pv. pruni were discovered: low copper tolerance (LCT; growth up to 38 μg/ml Cu 2+ ) and high copper tolerance (HCT; growth up to 51 μg/ml Cu 2+ ). A total of 26 (23 LCT and 3 HCT) out of 165 isolates in 2018 and 32 (20 LCT and 12 HCT) out of 133 isolates in 2019 were tolerant to copper. Peach leaves on potted trees were sprayed with copper rates typically applied at the stages of delayed dormancy (high rate; 2,397 μg/ml Cu 2+ ), shuck split (medium rate; 599 μg/ml Cu 2+ ), and during summer cover sprays (low rate; 120 μg/ml Cu 2+ ), and subsequently inoculated with sensitive, LCT, and HCT strains. Results indicated that the low and medium rates of copper reduced bacterial spot incidence caused by the sensitive strain but not by the LCT and HCT strains. This study confirms existence of X . arboricola pv. pruni tolerance to copper in commercial peach orchards in the southeastern United States, and suggests its contribution to bacterial spot development under current management practices.