Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes.

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager's activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

Interactions Among Heterodera glycines, Macrophomina phaseolina, and Soybean Genotype.

Heterodera glycines, the soybean cyst nematode (SCN), and fungal pathogen Macrophomina phaseolina are economically important soybean pathogens that may coinfest fields. Resistance remains the most effective management tactic for SCN, and the rhg1-b resistance allele derived from plant introduction 88788 is most commonly deployed in the northern United States. The concomitant effects of SCN and M. phaseolina on soybean performance, as well as the effect of the rhg1-b allele in two different genetic backgrounds, were evaluated in three environments (during 2013 to 2015) and a greenhouse bioassay. Within two soybean populations, half of the lines had the rhg1-b allele, and the other half had the susceptible allele in the backgrounds of the cultivars IA3023 and LD00-3309. Significant interactions between soybean rhg1-b allele and M. phaseolina-infested plots were observed in 2014. In all experiments, initial SCN populations (Pi) and M. phaseolina in roots were associated with reduced soybean yield. SCN reproduction factor (RF = final population/Pi) was affected by SCN Pi, rhg1-b, and genetic background. A background-by-genotype interaction on yield was observed only in 2015, with a stronger rhg1-b effect in the LD00-3309 background, which suggested that the susceptible parent 'IA3023' is tolerant to SCN. SCN female index from greenhouse experiments was compared with field RF, and Lin's concordance and Pearson's correlation coefficients decreased with increasing field SCN Pi in soil. In this study, both SCN and M. phaseolina reduced soybean yield asymptomatically, and the impact of SCN rhg1-b resistance was dependent on SCN virulence but also population density.

First report of foliar nematode (Aphelenchoides pseudobesseyi) on soybean in Colombia.

Soybean (Glycine max L.) is produced in over 70,000 ha in the Altillanura Region, eastern Colombia (Agronet 2023). From 2018 to 2020, foliar symptoms like green stem and foliar retention of soybean, which in Brazil can cause up to 100% soybean yield losses (Meyer et al. 2017), were observed in soybean fields in Colombia. During 2020, samples from symptomatic plants in reproductive stages (R1-R8) were collected from different commercial soybean fields in the Altillanura Region. Over 200 samples were processed, using an incubation method described in Coyne et al. (2014). Nematodes were recovered from photosynthetic leaf tissues and enlarged nodes/buds with population densities ranging from 13 to 132 and 36 to 936 nematodes/10g, respectively. Adult females were morphologically and molecularly characterized as Aphelenchoides pseudobesseyi (Oliveira et al. 2019; Subbotin et al. 2020). Female body length (n = 20) ranged from 653.3 to 806.3 µm (mean = 723 µm ± 52.7), stylet length from 11.0 to 12.3 µm (11.8 µm ± 0.3), body diameter from 14.8 to 17.9 µm (16.3 µm ± 1.1), post-uterine sac length from 38.7 to 51.9 µm (44.6 µm ± 5.1), vulva to anus from 145.5 to 223.2 µm (172.2 µm ± 22.4), and 26% of the vulva-anus distance. Genomic DNA was extracted (QIAGEN DNeasy® Blood & Tissue kit) from a pool of nematodes. The D2A/D3B (Tenente et al. 2004) primers were used to amplify and sequence the D2/D3 expansion region of the 28S rRNA gene. PCR product (~759 bp) was purified, sequenced, deposited in GenBank (OQ930285), and compared to previously deposited sequences (e.g., KX356756, KY510840, KY510839, KY510841, KT692694, KY510842, MH187565) by means of the BLAST algorithm. Similarly, 988F and 18SR-Burs (De Jesus et al. 2016) primers were used to amplify and sequence the near full-length 18S RNA gene (SSU). PCR product was purified, sequenced, deposited in GenBank (OQ954344), and compared to previously deposited sequences (e.g., KT454962, KT943534, KT943535, KY510835, KY510836, KY510837, KY510838, MH187565). Phylogenetic Bayesian analysis (Ronquist et al. 2012) of the of the D2/D3 and 18S regions placed this nematode from Colombia in the A. pseudobesseyi clade (PP = 100). To fulfill a modified Koch's postulates, the A. pseudobesseyi population described above was used in a greenhouse assay. In total, 120 soybean plants (cv. Flor Blanca) were infected with 200 A. pseudobesseyi (females + males)/plant. Briefly, at cotyledon stage (VC), 50 µl aliquot containing 50 A. pseudobesseyi was delivered onto each cotyledon and unifoliolate leaves (200 nematodes/plant). Sterile water was delivered to 80 plants which served as control. Plants were kept in the greenhouse at approximately 25°C and covered with clear plastic bag for 72 h to maintain over 90% relative humidity. After 15, 30, 45, and 60 days, soybean plants (n = 20) were processed, A. pseudobesseyi quantified, and the average reproduction factor (final population/initial population) was 0.1, 2.9, 14.0, and 1.8, respectively. Infected plants showed symptoms of blistering leaves with malformation (midrib vein twist), and A. pseudobesseyi was not observed in control plants. To our knowledge, this is the first report of A. pseudobesseyi parasitizing soybean buds and leaves in Colombia. Soybean is an important commodity for the Altillanura Region, and it is important to monitor the risk posed by this nematode. Furthermore, a better understanding of the nematode-host interaction and epidemiology in Colombia soybean producing regions is needed.

First report of Zymoseptoria tritici causing Septoria tritici blotch in wheat in Paraguay.

Wheat (Triticum aestivum) is the third most cultivated field crop in Paraguay; it is grown on over 450,000 hectares with an annual production of 927,776 tons (fao.org/faostat). In 1952, Septoria tritici blotch (STB) was associated with the fungus Septoria tritici solely based on microscopic observation of conidia (Viedma and Delgado 1987). However, no morphometric or molecular studies have been performed in Paraguay up to date. Over the following decades, STB epidemic outbreaks were recorded, with a reduction in wheat production of up to 70% (Viedma and Delgado 1987). During winter 2021, leaf blotch symptoms were observed with an incidence above 50% in wheat fields in Capitán Miranda, Itapúa, Paraguay. Scattered, spherical, buried, and light brown necrotic spots with dark edges were observed on the leaves. Pycnidia with prominent central ostiole were observed. Leaves with symptoms were washed with 1% sodium hypochlorite for 1 min, rinsed with sterile distilled water, and incubated in wet chambers to induce sporulation of the fungus. Pycnidia produced greyish to white cirri. Isolated conidia were thin, elongated, and hyaline, ranging from 26.9-72.7 × 1.5-2.9 µm with one to three septa. Monosporic colonies on potato dextrose agar (PDA, ; Difco laboratories, Detroit, MI) media varied in color from white to pink, dark gray to black, or black with stroma-like structures. Based on morphology, the fungus was characterized as Zymoseptoria tritici (Hoorne et al. 2002; Gilchrist-Saavedra et al. 2005). Fungal DNA was extracted from mycelia, and the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), 28S rRNA gene (LSU), actin gene (act), calmodulin (CaM) were amplified using ITS1/ITS4, EF1-728F/EF-2, LSU1Fd/ LR5, ACT-512F/ACT-783R, CAL-228F/CAL737R primers, respectively. PCR amplicons were sequenced at Macrogen (Seoul, Republic of Korea) and deposited in the NCBI GenBank database (ITS: OQ360718; TEF1-α: OQ999044, LSU: OQ996413, act: OQ999046, CaM: OQ999045). Sequences were aligned with several isolates of Septoria spp. previously reported (Verkley et al. 2013; Stukenbrock et al. 2012) using ClustalW. The alignments were concatenated with Bioedit (Hall 1999). The UPGMA method with 1,000 bootstrap replications, was used to construct the phylogenetic tree using MEGA11 with Readeriella mirabilis as the outgroup. The isolate from Paraguay grouped into the Zymoseptoria tritici clade with 96% bootstrap support. To confirm pathogenicity, ten wheat plants cv. Itapúa 80 were grown in pots for three weeks in growth chambers (22 ± 2°C; 16 h photoperiod). Subsequently, these plants were inoculated with 1×107 conidia ml-1 suspension, and ten non-inoculated plants served as control. Seven days after inoculation (DAI), symptoms were observed displaying oval necrotic lesions and approximately 14 DAI abundant pycnidia were observed on and around the lesions. Segments of symptomatic leaves were placed in moisture chambers overnight to enhance cirri development. Conidia were mounted on a slide and observed under the compound microscope. Individual cirrhus were transferred to plates containing PDA and produced colonies like those used in the inoculation (Hoorne et al. 2002). We confirmed that the causal agent of STB from wheat fields in Paraguay was Zymoseptoria tritici. This pathogen causes annual wheat disease epidemics in Paraguay; therefore, optimizing surveillance for early detection and understanding its distribution will improve integrated management.

First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay.

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 µm; truncated with protruding hilum and varied in length from 1.0 to 1.5 µm and diameters from 2.0 to 2.2 µm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

Incidence, Population Density, and Spatial Heterogeneity of Plant-Parasitic Nematodes in Corn Fields in Ohio.

Soil samples were collected from 425 corn fields in 28 Ohio counties between growth stages V3 and V6 during the 2013 and 2014 growing seasons. Ten morphological groups of plant-parasitic nematodes, namely spiral, lesion, lance, dagger, stunt, pin, ring, stubby-root, cyst, and "tylenchids" (several genera morphologically similar to members of the subfamily Tylenchinae [NCBI Taxonomy] including Cephelenchus, Filenchus, Malenchus, and Tylenchus) were identified. Eight species belonging to six of these groups were characterized. Spiral, tylenchids, lesion, pin, lance, stunt, and dagger nematodes were detected in 94, 96, 80, 57, 48, 48, and 37% of the fields, respectively, whereas the stubby-root, cyst, and ring nematodes were present in fewer than 14% of the samples. Averaged across fields, the spiral, tylenchids, and pin nematodes had the highest mean population densities. For all groups, incidence and population density varied among counties, and in some cases, among soil regions and cropping practices. Both population parameters were heterogeneous at multiple spatial scales, with the lowest heterogeneity among soil regions and the highest among fields within county and soil region. Estimated variances at the soil region level were not significantly different from zero for most of the nematodes evaluated. Stunt and lance were two of the most variable groups at all tested spatial scales. In general, the population densities were significantly more heterogeneous at the field level than at the county level. Findings from this study will be useful for developing sampling protocols and establishing on-farm trials to estimate losses and evaluate nematode management strategies.

Cropping Practices and Soil Properties Associated with Plant-Parasitic Nematodes in Corn Fields in Ohio.

Ten morphological groups of plant-parasitic nematodes (spiral, lesion, lance, dagger, stunt, pin, ring, stubby-root, cyst, and miscellaneous tylenchids) were detected in corn fields in Ohio, but the presence and population density of these groups varied among fields. Binary and ordinal logistic regression models were fitted to the data to estimate the odds of each group being present, and the lesion, lance, spiral, and pin nematode population densities being at moderate-high risk levels based on soil region, cropping sequence, tillage, and soil pH, silt content, and electrical conductivity. All covariates were associated with at least one nematode group, but soil region had the greatest and most consistent effect. Dagger and ring nematodes were more likely to be present in region 6 than in any of the other regions, whereas lance, stunt, pin, stubby-root, and spiral nematodes were more likely to be present in regions 1 to 5 than 6. Spiral, lance, and pin nematode population densities were more likely to be at moderate-high risk levels in regions 3 and 4 than in region 6. Fields under conservation tillage were two times more likely to have moderate-high risk lance nematode population densities than fields under conventional tillage. Similarly, pin nematode population densities were two times more likely to be at moderate-high risk levels in fields under rotation than in continuous corn. For every unit increase in soil pH, the odds of the spiral nematode population density being at moderate-high risk levels increased by 43%, but the odds of the lesion and pin nematode population densities being at the same risk level decreased by 63 and 29%, respectively. The predicted probability of lesion and lance population densities being at moderate-high risk levels decreased as the silt content of the soil increased. These finding will be useful for developing future nematode sampling protocols and for assessing the risk associated with nematodes in corn fields in Ohio.

Evaluation of Cultivar Resistance to Soybean Cyst Nematode with a Quantitative Polymerase Chain Reaction Assay.

Heterodera glycines, the soybean cyst nematode, is a major pathogen of soybean. Effective management of this pathogen is contingent on the use of resistant cultivars; thus, screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative polymerase chain reaction (qPCR). This method will serve as a prelude to differentiation of resistance levels in soybean cultivars. A reproducible inoculation method was developed by means of a sand column to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours after infestation, the roots were surface-sterilized and genomic DNA (gDNA) was extracted. For the qPCR assay, a primer pair for the single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines gDNA amplification within soybean roots. Compatible 'Lee 74', incompatible 'Peking', and cultivars with different levels of resistance to H. glycines were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours postinfestation, infected seedlings were transplanted into pasteurized soil. Subsequently, they were harvested at 1, 7, 10, 14, and 21 days postinfestation for gDNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice. This qPCR assay has the potential to replace the traditional Female Index-based screening and improve precision in determining infection levels.

Trichothecene Genotype Profiling of Wheat Fusarium graminearum Species Complex in Paraguay.

Paraguay is a non-traditional wheat-producing country in one of the warmest regions in South America. Fusarium Head Blight (FHB) is a critical disease affecting this crop, caused by the Fusarium graminearum species complex (FGSC). A variety of these species produce trichothecenes, including deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). This study characterized the phylogenetic relationships, and chemotype diversity of 28 strains within FGSC collected from wheat fields across different country regions. Phylogenetic analysis based on the sequence of elongation factor-1α gene (EF-1α) from 28 strains revealed the presence of four species in the FGSC: F. graminearum sensu stricto, F. asiaticum, F. meridionale and F. cortaderiae. Ten strains selected for further analysis revealed that all F. graminearum strains were 15-ADON chemotype, while the two strains of F. meridionale and one strain of F. asiaticum were NIV chemotype. Thus, the 15-ADON chemotype of F. graminearum sensu stricto was predominant within the Fusarium strains isolated in the country. This work is the first report of phylogenetic relationships and chemotype diversity among Fusarium strains which will help understand the population diversity of this pathogen in Paraguay.

Plant-Parasitic Nematodes Are Potential Pathogens of Miscanthus × giganteus and Panicum virgatum Used for Biofuels.

A survey of Miscanthus × giganteus and switchgrass plots throughout the midwestern and southeastern United States was conducted to determine the occurrence and distribution of plant-parasitic nematodes associated with these biofuel crops. During 2008, rhizosphere soil samples were collected from 24 Miscanthus × giganteus and 38 switchgrass plots in South Dakota, Iowa, and Illinois. Additional samples were collected from 11 Miscanthus × giganteus and 10 switchgrass plots in Illinois, Kentucky, Georgia, and Tennessee the following year. The 11 dominant genera recovered from the samples were Pratylenchus, Helicotylenchus, Xiphinema, Longidorus, Heterodera, Hoplolaimus, Tylenchorhynchus, Criconemella, Paratrichodorus, Hemicriconemoides, and Paratylenchus. Populations of Helicotylenchus, Xiphinema, and Pratylenchus were common and recorded in 90.5, 83.8, and 91.9% of the soil samples from Miscanthus × giganteus, respectively, and in 91.6, 75, and 83.3% of the soil samples from switchgrass, respectively. Prominence value (PV) (PV = population density × âˆšfrequency of occurrence/10) was calculated for the nematodes identified. Helicotylenchus had the highest PV (PV = 384) and was followed by Xiphinema (PV = 152) and Pratylenchus (PV = 72). Several of the nematode species associated with the two biofuels crops were plant parasites. Of these, Pratylenchus penetrans, P. scribneri, P. crenatus, Helicotylenchus pseudorobustus, Hoplolaimus galeatus, X. americanum, and X. rivesi are potentially the most damaging pests to Miscanthus × giganteus and switchgrass. Due to a lack of information, the damaging population thresholds of plant-parasitic nematodes to Miscanthus × giganteus and switchgrass are currently unknown. However, damage threshold value ranges have been reported for other monocotyledon hosts. If these damage threshold value ranges are any indication of the population densities required to impact Miscanthus × giganteus and switchgrass, then every state surveyed has potential for yield losses due to plant-parasitic nematodes. Specifically, Helicotylenchus, Xiphinema, Pratylenchus, Hoplolaimus, Tylenchorhynchus, Criconemella, and Longidorus spp. were all found to have population densities within or above the threshold value ranges reported for other monocotyledon hosts.