First report of Fusarium sporotrichioides causing Fusarium fruit rot on peaches in Pakistan.

Peach (Prunus persica) is an important stone fruit crop cultivated on 14,350 hectares in temperate areas of Pakistan, where post-harvest fungal rots cause major economic losses for growers. During April-August 2019, rotted peaches were observed in fruit markets of Rawalpindi district (33° 37' 33.8052'' N and 73° 4' 17.1912'' E), Punjab Province, Pakistan. Symptomatic fruit were surface sterilized for 3 min in 0.5% NaClO and 1 min in 70% ethanol, then rinsed three times in sterilized distilled water and air-dried for 30 minutes on filter paper. Tissue segments were excised from the margins of rot lesions and placed on potato dextrose agar (PDA), followed by incubation for one week at 25 +1o C. Furthermore, to maintain original sporodochial morphology and conidial description tissue segments were also grown on carnation leaf agar (CLA) and synthetic nutrient agar (SNA) (Sever et al., 2012). A total of 22 isolates were obtained from different fruit markets; colonies were fast-growing with pinkish-white, fluffy mycelia. Microconidia were abundant, 7.5 to 11.3 × 2.1 to 4.1 µm, oval to spindle-shaped, thin-walled, and hyaline whereas, macroconidia were thick-walled, sickle-shaped, 5- to 7-septate, hyaline, and 23.5 to 39.7 × 3.7 to 7.0 µm on carnation leaf agar. Virulence intensity of fungal isolates was confirmed after pathogenicity. Based on morphological characteristics, isolates were identified provisionally as Fusarium sporotrichioides (Leslie and Summerell 2006). For molecular identification, three target gene regions (ITS, elongation factor (EF), and ß-tubulin benA) were amplified for highly pathogenic fungal isolates (FUS21K, FUS9SM, and FUS63KP) (O'Donnell et al. 2015). BLAST searches of the obtained sequences indicated 100% homology with GenBank accession number MN452643, and 99.56% homology with MT635298 for the ITS region, similarly 100% identity with GenBank accession numbers MN555016, MN555126, and MN555037 for the EF gene, and 100% homology with GenBank accession numbers MK435577, MH791363, and GQ915448 for ß-tubulin benA of the F. sporotrichioides species complex (O'Donnell et al. 2015). Sequences were deposited in GenBank; accession numbers for each gene were as follows: ITS, ON180670, ON180671, and ON180672; EF, ON312093, ON312094, and ON312095; and ß-tubulin benA, ON312090, ON312091, and ON312092. Maximum likelihood analysis on RPB1 and RPB2 nucleotide sequences of various fusaria was conducted which revealed that F. sporotrichioides formed a monophyletic relationship to terminal fusarium clade (TFC) comprising 22 strongly supported species complexes and 10 monotypic lineages, which were provisionally recognized as Fusarium. To complete Koch's postulates, 10µl aliquots of spore suspensions (106 spores/ml) of each FUS21K, FUS9SM and FUS63KP were pipetted individually onto six ripe, asymptomatic peaches. Sterile distilled water was applied as a negative control on four fruit. Fruit were incubated at 25 ± 1°C for one week in a sterile moist chamber, and the trial was conducted three times. Rot symptoms associated with pinkish-white fluffy mycelia were observed on inoculated fruits after 72 hours, whereas no mycelium was observed on the negative controls. The cultures on PDA, CLA and SNA from each of the inoculated fruit were morphologically identical to the original culture. The pathogen was confirmed as a member of F. sporotrichioides species complex based on pinkish-white mycelium, with red color on the underside of petri plates and the manner in which conidia are born on conidiophores. This is the first report of F. sporotrichioides on peaches from Pakistan, providing an important foundation for peach growers, agricultural advisers, plant pathologists, and plant breeders to improve the management of this economically damaging disease.

Occurrence in Seeds and Potential Seed Transmission of Xanthomonas vasicola pv. vasculorum in Maize in the United States.

This paper reports original evidence regarding the potential role of seed transmission of Xanthomonas vasicola pv. vasculorum in the epidemiology of bacterial leaf streak (BLS) in maize. We evaluated the occurrence of the pathogen on seeds from diseased fields and its subsequent transmission to seedlings. In 2016 and 2017, X. vasicola pv. vasculorum was detected by TaqMan PCR from 22 of 41 maize seed lots harvested from naturally infected fields in Colorado, Nebraska, and Iowa. However, many of the PCR-positive samples did not yield culturable X. vasicola pv. vasculorum colonies. The highest levels of seed contamination were detected in dent maize and popcorn from NE and CO. Seed transmission was evaluated in greenhouse grow-outs from eight seed lots, totaling more than 14,000 plants. Putative seed transmission events from naturally contaminated seed lots, estimated from PCR results, occurred at a frequency between 0.1 and 0.5% in 10-seedling pooled samples and at a frequency of 2.7% from individual plant assays. However, no seedling symptoms were observed during these assays and live X. vasicola pv. vasculorum colonies were not recovered from PCR-positive seedlings. In contrast, seed transmission was readily demonstrated from artificially contaminated seed lots, including typical symptoms and recovery of live bacteria. Seed transmission consistently occurred from seeds soaked in bacterial suspensions with concentrations of ≥106 CFU/ml, suggesting that a threshold population of the bacterium is necessary for the development of BLS symptoms and recovery of live bacteria. The low bacterial populations on naturally contaminated seeds apparently were not sufficient to result in diseased seedlings.

Evaluating the Performance of a Relative Humidity-Based Warning System for Sooty Blotch and Flyspeck in Iowa.

A warning system for the sooty blotch and flyspeck (SBFS) fungal disease complex of apple, developed originally for use in the southeastern United States, was modified to provide more reliable assessment of SBFS risk in Iowa. Modeling results based on previous research in Iowa and Wisconsin had suggested replacing leaf wetness duration with cumulative hours of relative humidity (RH) ≥97% as the weather input to the SBFS warning system. The purpose of the present study was to evaluate the performance of a RH-based SBFS warning system, and to assess the potential economic benefits for its use in Iowa. The warning system was evaluated in two separate sets of trials-trial 1 during 2010 and 2011, and trial 2 during 2013-2015-using action thresholds based on cumulative hours of RH ≥97% and ≥90%, respectively, in conjunction with two different fungicide regimes. The warning system was compared with a traditional calendar-based system that specified spraying at predetermined intervals of 10 to 14 days. In trial 1, use of the RH ≥97% threshold caused substantial differences between two RH sensors in recording number of hours exceeding the threshold. When both RH thresholds were compared for 2013-2015, on average, RH ≥90% resulted in a 53% reduction in variation of cumulative hours between two identical RH sensors placed adjacent to each other in an apple tree canopy. Although both the SBFS warning system and the calendar-based system resulted in equivalent control of SBFS, the warning system required fewer fungicide sprays than the calendar-based system, with an average of 3.8 sprays per season (min = 2; max = 5) vs. 6.4 sprays per season (min = 5; max = 8), respectively. The two fungicide regimes provided equivalent SBFS control when used in conjunction with the warning system. A partial budget analysis showed that using the SBFS warning system with a threshold of RH ≥90% was cost effective for orchard sizes of >1 ha. The revised warning system has potential to become a valuable decision support tool for Midwest apple growers because it reduces fungicide costs while protecting apples as effectively as a calendar-based spray schedule. The next step toward implementation of the SBFS warning system in the North Central U.S. should be multiyear field testing in commercial orchards throughout the region.

Roles of Genotype-Determined Mycotoxins in Maize Seedling Blight Caused by Fusarium graminearum.

Fusarium graminearum is an important causal agent of maize seedling blight. The species includes several chemotypes that produce various forms of deoxynivalenol (DON) and nivalenol (NIV). To understand the effects and roles of F. graminearum mycotoxins on maize seedling blight occurring at Zhang Ye of Gansu, China, 23 isolates of F. graminearum were collected and characterized. A PCR assay showed all 23 isolates belonged to the 15-acetyldeoxynivalenol (15-ADON) genotype. This was also confirmed by production of both DON and 15-ADON in either rice culture medium or maize seedling roots, detected by high performance liquid chromatography and mass spectrometry. In maize seedling roots, 15-ADON dominated at 6 days post inoculation (dpi) and DON was the main mycotoxin at 12 dpi. The biomass of F. graminearum doubled from 6 to 12 dpi, and was positively correlated with virulence of the isolates. Both mycotoxins affected maize root vitality, but 15-ADON had a greater effect than DON. ALDH9 and MDH, two dehydrogenase synthesis genes in maize, showed a lower relative expression in 15-ADON treatments than in DON treatments. It indicated that both mycotoxins affected seed germination and root development, with 15-ADON being more destructive. Under scanning electron microscopy and transmission electron microscopy, root hair formation and development were delayed by DON, but completely inhibited by 15-ADON. 15-ADON caused cell shrinkage, loose cellular structure, and widened intercellular spaces; it also destroyed organelles and caused plasmolysis, and eventually ruptured cell membranes causing cell death. DON did not affect cell morphology and arrangement, but altered the morphology of organelles, forming concentric membranous bodies and a large amount of irregular lipid droplets. Thus, both mycotoxins contributed to symptom expression of maize seedling blight, but 15-ADON was more destructive than DON.