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Optimization of oncolytic viruses for therapeutic applications requires the strategic removal or mutagenesis of virulence genes alongside the insertion of transgenes that enhance viral replication, spread and immunogenicity. However, the complexity of many viral genomes and the labour-intensive nature of methods for the generation and isolation of recombinant viruses have hindered the development of therapeutic oncolytic viruses. Here we report an iterative strategy that exploits the preferential susceptibility of viruses to certain antibiotics to accelerate the engineering of the genomes of oncolytic viruses for the insertion of immunomodulatory cytokine transgenes, and the identification of dispensable genes with regard to replication of the recombinant oncolytic viruses in tumour cells. We applied the strategy by leveraging insertional mutagenesis via the Sleeping Beauty transposon system, combined with long-read nanopore sequencing, to generate libraries of herpes simplex virus type 1 and vaccinia virus, identifying stable transgene insertion sites and gene deletions that enhance the safety and efficacy of the viruses., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Devastating disease outbreaks with leaf spot symptoms and fruit rot caused by a fungus identified as a cryptic species of Neopestalotiopsis have generated concern in the Florida strawberry industry. Some Neopestalotiopsis species are saprobes in soil and plant debris, but the ability of the new Neopestalotiopsis sp. to survive in strawberry debris was unknown. Therefore, the objectives of this study were to develop a semiselective medium for isolation of Neopestalotiopsis spp. and to evaluate Neopestalotiopsis sp. survival in soil and strawberry plant debris over the summer in Florida. The Neopestalotiopsis semiselective medium (NSM) inhibited growth of most fungal species pathogenic to strawberry in Florida, except Neopestalotiopsis rosae , Neopestalotiopsis sp., and Phomopsis obscurans , in addition to Penicillium spp., which are nonpathogenic. However, Neopestalotiopsis species, P . obscurans , and Penicillium spp. could be distinguished in the medium through morphological characteristics. Soil samples arbitrarily collected from six commercial fields toward the end of the season (May) and before (July) and after (September) preplant soil fumigation in the following season were processed with NSM, and Neopestalotiopsis spp. populations were calculated as CFU per gram of soil. CFU ranged from 48.3 to 2,410.8 at the end of the season and from 1.7 to 630.8 before soil fumigation, but Neopestalotiopsis spp. were not recovered after soil treatment. However, 1.7 to 25 CFU were obtained from the nontreated areas in the row middles. Neopestalotiopsis sp. survival was also evaluated on diseased strawberry plants (leaves and crowns) exposed to the environment for 17 months. On leaves, Neopestalotiopsis spp. were recovered for 6 months before leaf decomposition, whereas the number of CFU on crowns declined monthly, but colonies were still recovered during the final evaluation months. A high-resolution melting analysis confirmed most of the colonies from soil and crowns were the new Neopestalotiopsis sp. Our results show Neopestalotiopsis sp. can survive in soil and strawberry debris under Florida summer conditions and may serve as a source of inoculum for the subsequent season., Competing Interests: The author(s) declare no conflict of interest.

Succinate dehydrogenase inhibitors (SDHIs) are the fungicides most commonly used to control Botrytis fruit rot on commercial strawberry in Florida. The medium-to-high risk of selection of resistance in the causal agent Botrytis cinerea is a threat to the efficacy of this fungicide group. In this study, we characterized the sensitivity of B. cinerea to the SDHI isofetamid, evaluated the SdhB gene mutation associated with resistance, and monitored resistance frequencies to five SDHI fungicides for two consecutive seasons. EC 50 values of 70 isolates were obtained using the spiral gradient dilution (SGD) method and averaged 0.098 µg/ml of isofetamid. EC 50 averages of 3.04 and >500.00 µg/ml were obtained for isolates with the N230I and P225F mutations indicating moderate and high resistance to isofetamid, respectively. A total of 565 B. cinerea isolates collected during 2015-2016 and 2016-2017 seasons from strawberry nurseries and Florida production fields were evaluated using conidial germination assays. Results for the first season showed resistance frequencies of 95, 33, 21, 25, and 0% to boscalid, penthiopyrad, fluopyram, benzovindiflupyr, and isofetamid, respectively. The respective resistance frequencies for the following season were 91, 95, 44, 27, and 1.3%. Only three isolates were found to be moderately resistant to isofetamid during the second season, and the mutation N230I was identified after sequence analysis. These isolates were confirmed to be resistant to isofetamid in fruit assays with disease incidence of 55.6 to 77.0%; however, the conidial production of the isolates was inhibited by an average of 83.9%. In general, isofetamid efficacy was higher than the other evaluated SDHIs, but a slight increase in resistance frequencies was observed in our study.

Succinate dehydrogenase inhibitors (SDHIs) are an essential group of fungicides for managing gray mold, caused by Botrytis cinerea , in numerous crops. Resistance to boscalid, an early-generation SDHI, is widespread worldwide and was linked to mutations in the iron-sulfur protein encoding the SdhB subunit of the SDH complex. Herein, we report on four simultaneous dependent mutations at codons 85 (G85A), 93 (I93V), 158 (M158V), and 168 (V168I) of the membrane-anchored SdhC subunit of B. cinerea . Isolates without and with mutations in SdhC were referred to as C - and C + genotypes, respectively. The C + genotype was found in all the five surveyed hosts from different U.S. regions but its frequency was higher, 25 to 40%, in the tree fruit isolates compared with 12 to 25% in the small fruit populations. The four SdhC mutations were found in isolates without mutations in SdhB or with mutations known to confer resistance to the SDHIs in SdhB. However, the frequency of C + isolates was significantly higher in the SdhB wild-type isolates, which suggests that SDHI sprays may have played a role in selecting for the C - over the C + genotype. Field C + isolates exhibited reduced sensitivity to fluopyram and increased sensitivity to boscalid and penthiopyrad in vitro and on detached fruit. Homology modeling confirmed the positioning of the four mutations in the ubiquinone-binding pocket. The SdhC G85A is found in the proximal ubiquinone binding site and SdhC M158V is positioned in the iron sulfur protein interface next to the [3Fe-4S] cluster, whereas SdhC I93V is positioned next to the heme b with vital functions in the SDH enzyme. Beside the differential sensitivity to the SDHIs, these mutations caused a significant fitness cost in the C + isolates including sporulation and increased sensitivity to reactive oxygen species. The presence of Botrytis populations differentially sensitive to the SDHIs suggests increased risks for resistance development but also opens up new perspective for future gray mold management using different SDHI fungicides.

Botrytis fruit rot (BFR), caused by the necrotrophic fungus Botrytis cinerea, is the most important disease of strawberry and is mainly controlled by applications of fungicides from multiple chemical groups. To develop more effective and sustainable BFR management programs, field trials were conducted to evaluate the efficacy of fluopyram and penthiopyrad, two newly registered succinate dehydrogenase inhibitors (SDHIs), rotated or tank mixed with the multisites thiram and captan or the single-sites fludioxonil and fenhexamid. The treatments were applied at two different strawberry fields during the 2013-14 and 2014-15 seasons. Overall, tank mixtures of fluopyram and penthiopyrad increased yield and reduced BFR better than rotations with the same fungicides. The multisite thiram tank mixed with fluopyram reduced BFR incidence by 63 to 86% versus 56 to 84% when the two fungicides were rotated. Thiram tank mixed with penthiopyrad reduced BFR incidence by 55 to 72% versus 42 to 66% when rotated. Captan rotated or tank mixed with fluopyram had a positive effect on yield and BFR incidence, whereas the combination of captan with penthiopyrad had negative impacts. Similarly, the single-site fenhexamid had significant positive impacts when rotated or tank mixed with fluopyram but resulted in poor BFR control when combined with penthiopyrad. The rotation of fludioxonil with both SDHIs had a significant positive effect, although its combination with fluopyram was more effective. The multirotation consisting of both SDHIs and different multi- and single-site fungicides did not provide a greater efficacy than the dual rotation or tank-mixture programs. Our findings suggest more scrutiny is needed when recommending tank-mixture or rotation partners for new fungicides to ensure compatibility and enhanced BFR management. Future recommendations should emphasize the importance of such selections at an early stage for delaying fungicide resistance development and extending the lifespan of at-risk fungicides.

Incidence of blossom blight and Botrytis fruit rot (BFR), caused by Botrytis cinerea, on two southern highbush blueberry cultivars was evaluated in several blueberry fields grown in the vicinity (BB-Str (+) ) or not (BB-Str (-) ) of strawberry fields in central Florida. Blossom blight and BFR incidence were higher in BB-Str (+) fields in 2014 and significantly higher in 2015 compared to BB-Str (-) fields. In total, 613 B. cinerea isolates (i.e., 181 and 432 isolates from BB-Str (-) and BB-Str (+) fields, respectively) were collected. The isolates were evaluated for sensitivity to eight single-site and one multisite fungicides using a spore germination and a germ tube elongation assay. Overall, 5, 15, 24, 28, 54, and 93% of isolates collected from BB-Str (-) were resistant to penthiopyrad, cyprodinil, boscalid, fenhexamid, pyraclostrobin, and thiophanate-methyl, respectively. Respective resistance frequencies in BB-Str (+) isolates were 10, 30, 65, 66, 89, and 99%. Resistance frequencies for all fungicides were always higher in BB-Str (+) fields compared to BB-Str (-) fields. Isolates exhibiting resistance to six or five fungicides simultaneously were predominant (50 to 70%) in blueberry fields regardless if they were grown in the vicinity of strawberry fields or not. Among 308 and 305 B. cinerea isolates tested in 2014 and 2015, 41.8 and 47.1%, respectively, showed reduced sensitivity to the multisite fungicide captan. The lower label rate of captan applied preventively did not control isolates with reduced sensitivity on detached blueberry fruit. These findings suggest a potential population flow between strawberry and blueberry fields that may impact blossom blight and gray mold development in blueberry fields. The relatively lower fungicide input applied to blueberry fields compared with strawberry fields seems to be sufficient to select for resistance and multiple-resistant phenotypes in B. cinerea populations in blueberry.

Botrytis isolates from strawberry transplants originating from Canada and the northern United States as well as isolates collected from strawberry and blueberry commercial fields in the southeastern United States were investigated for the frequency of Botrytis cinerea, other cryptic Botrytis spp. reported recently, and the transposable elements (TE) using six genetic markers. B. cinerea sensu stricto was predominant (94%) in strawberry and blueberry in all surveyed regions. Botrytis group S, a newly reported clade on strawberry from Germany, was found at low frequencies (6%) in strawberry in the United States and Canada and on blueberry isolates from Florida. Neither B. caroliniana nor B. pseudocinerea were detected in the U.S. or Canadian populations. Transposa isolates containing the TE boty and flipper accounted for 74% of 410 isolates studied herein. Isolates containing boty only or flipper only elements were found at 21 and 2%, respectively. However, boty isolates were predominant in the blueberry population with more than 50%. The TE were found in B. cinerea and Botrytis group S at similar frequencies, except that flipper was more frequent (10.7%) in Botrytis group S, compared with 1.6% in B. cinerea. The sensitivity of 256 Botrytis isolates from the different genetic groups described above was evaluated to seven fungicides registered to control gray mold in commercial fields. Results indicate that B. cinerea and transposa isolates have higher resistance frequencies to almost all fungicides tested compared with the other Botrytis genotypes or species, whereas the TE flipper may be related to resistance to fludioxonil. Similarities observed between nursery and commercial field populations and their impact on gray mold development and management are discussed.

Strawberry transplants produced in nurseries across Canada, northern United States, and California are shipped annually to other strawberry-growing regions, including Florida. Botrytis cinerea, the causal agent of gray mold, causes latent infections on transplants which are suggested as a potential source of primary inoculum in strawberry fields. In this study, we investigated the survival of B. cinerea isolates over the summer in Florida, the presence of B. cinerea in transplants from 14 nurseries from Canada and the United States in 2011, 2012, and 2013, and the sensitivity of nursery population to several botryticides. Botrytis cinerea was detected on dead strawberry plants sampled from commercial strawberry fields between March and June but not in July and August, suggesting that the fungus does not over-summer in strawberry fields in Florida. Nursery transplants surveyed in 2011, 2012, and 2013 showed B. cinerea incidences of 20 to 37, 20 to 83, and 2.5 to 92.5%, respectively. In total, 409 isolates were tested for sensitivity to pyraclostrobin, boscalid, pyrimethanil, fenhexamid, iprodione, penthiopyrad, fluopyram, and fludioxonil. Overall, respective resistance frequencies were 91.7, 79.3, 33.2, 20.7, 2.4, 0.2, 0.2, and 0.0%. A majority of isolates tested were resistant to either 3 or 4 fungicides simultaneously. These findings reinforce the need for an integrated approach between strawberry nurseries and production fields to improve gray mold management and mitigate future risks of resistance development in B. cinerea.

Botryotinia fuckeliana de Bary (anamorph Botrytis cinerea Pers.) is an ubiquitous plant pathogen causing gray mold disease on more than 200 crops grown in the field or in greenhouses. Eucalyptus seedlings originating from three different greenhouses showing stem lesions were submitted to the Gulf Coast Research and Education Center Disease Clinic in June 2012. Ten single spore isolates of B. cinerea were obtained and tested for sensitivity using spore germination and germ tube elongation assays described previously (4). Fungicides tested were pyraclostrobin at 100 μg/ml (Cabrio, BASF, Research Triangle Park, NC), thiophanate-methyl at 100 μg/ml (Topsin-M, UPI, King of Prussia, PA), fenhexamid at 1 and 50 μg/ml (Elevate, Arysta Life Sciences, Cary, NC), fludioxonil at 0.1 and 10 μg/ml (Medallion, Syngenta Crop Protection, Research Triangle Park, NC), and iprodione at 5 and 50 μg/ml (Rovral, Bayer CropScience, Greensboro, NC) on 1% malt extract agar (MEA, 10 g malt extract and 15 g agar), and to cyprodinil at 1 and 25 μg/ml (Vanguard, Syngenta Crop Protection) on 0.5% sucrose agar (4). Sensitivity to the succinate dehydrogenase inhibitors (SDHIs) boscalid at 5 μg/ml (Endura, BASF), penthiopyrad at 1 and 3 μg/ml (Fontelis, DuPont Crop Protection, Willington, DE), and fluopyram at 3 μg/ml (Luna Privilege, Bayer CropScience) was evaluated on yeast bacto acetate agar (YBA) (3). The discriminatory dose for boscalid was adapted from (2) whereas those used for penthiopyrad and fluopyram were developed in this study. Isolates were grown on malt yeast extract agar for 7 to 10 days and spore suspensions were prepared in sterile distilled water and diluted to 10 6 conidia/ml. Respective media in 9-cm petri dishes were seeded with 7-μl droplets from each isolate allowing testing for all isolates on one plate. Two plates were used for each fungicide and sensitivity tests were repeated twice. Germination and germ tube growth were assessed microscopically after 16 to 24 h incubation at 22°C. The frequency of isolates resistant to two, three, and four fungicides was 90, 60, and 10%, respectively. Nine isolates (90%) were resistant to thiophanate-methyl and pyraclostrobin, simultaneously, whereas six (60%) and two isolates (20%) were resistant to boscalid and fenhexamid, respectively. All boscalid-resistant isolates were also resistant to pyraclostrobin and thiophanate-methyl, but one fenhexamid-resistant isolate was sensitive to the other three fungicides. Eight isolates that germinated at 5 μg/ml iprodione but not at 50 μg/ml were considered sensitive. All isolates were sensitive to the SDHIs penthiopyrad and fluopyram as well as to cyprodinil and fludioxonil. To our knowledge, this is the first report of resistance to pyraclostrobin, thiophanate-methyl, fenhexamid, and boscalid in B. cinerea from eucalyptus seedlings in Florida. The absence of resistance to fludioxonil and iprodione is likely because these fungicides are not registered in nurseries as well as fluopyram and penthiopyrad which were developed only recently. Management practices should be developed to limit the selection and spread of additional resistant populations in eucalyptus nurseries as has occurred in Florida strawberries where multi-fungicide resistance is widespread (1). References: (1) A. Amiri et al. Plant Dis. 97:393, 2013. (2) M. Leroch et al. Appl. Environ. Microbiol. 79:159, 2013. (3) G. Stammler and J. Speakman. J. Phytopathol. 154:508, 2006. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.