Viruses of Apple Are Seedborne but Likely Not Vertically Transmitted.

Many viruses occur in apple (Malus domestica (Borkh.)), but no information is available on their seed transmissibility. Here, we report that six viruses infecting apple trees, namely, apple chlorotic leaf spot virus (ACLSV), apple green crinkle-associated virus (AGCaV), apple rubbery wood virus 2 (ARWV2), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and citrus concave gum-associated virus (CCGaV) occur in seeds extracted from apple fruits produced by infected maternal trees. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) assays revealed the presence of these six viruses in untreated apple seeds with incidence rates ranging from 20% to 96%. Furthermore, ASPV was detected by RT-PCR in the flesh and peel of fruits produced by infected maternal trees, as well as from seeds extracted from apple fruits sold for fresh consumption. Finally, a large-scale seedling grow-out experiment failed to detect ACLSV, ASGV, or ASPV in over 1000 progeny derived from sodium hypochlorite surface sterilized seeds extracted from fruits produced by infected maternal trees, suggesting no detectable transmission via embryonic tissue. This is the first report on the seedborne nature of apple-infecting viruses.

Pyricularia Are Mostly Host-Specialized with Limited Reciprocal Cross-Infection Between Wheat and Endemic Grasses in Minas Gerais, Brazil.

Wheat blast, caused by Pyricularia oryzae Triticum (PoT), is an emerging threat to global wheat production. The current understanding of the population biology of the pathogen and epidemiology of the disease has been based on phylogenomic studies that compared the wheat blast pathogen with isolates collected from grasses that were invasive to Brazilian wheat fields. In this study, we performed a comprehensive sampling of blast lesions in wheat crops and endemic grasses found in and away from wheat fields in Minas Gerais. A total of 1,368 diseased samples were collected (976 leaves of wheat and grasses and 392 wheat heads), which yielded a working collection of 564 Pyricularia isolates. We show that, contrary to earlier implications, PoT was rarely found on endemic grasses, and, conversely, members of grass-adapted lineages were rarely found on wheat. Instead, most lineages were host-specialized, with constituent isolates usually grouping according to their host of origin. With regard to the dominant role proposed for signalgrass in wheat blast epidemiology, we found only one PoT member in 67 isolates collected from signalgrass grown away from wheat fields and only three members of Urochloa-adapted lineages among hundreds of isolates from wheat. Cross-inoculation assays on wheat and a signalgrass used in pastures (U. brizantha) suggested that the limited cross-infection observed in the field may be due to innate compatibility differences. Whether or not the observed level of cross-infection would be sufficient to provide an inoculum reservoir, or serve as a bridge between wheat growing regions, is questionable and, therefore, deserves further investigation.

Identification and high-throughput genotyping of single nucleotide polymorphism markers in a non-model conifer (Abies nordmanniana (Steven) Spach).

Single nucleotide polymorphism (SNP) markers are powerful tools for investigating population structures, linkage analysis, and genome-wide association studies, as well as for breeding and population management. The availability of SNP markers has been limited to the most commercially important timber species, primarily due to the cost of genome sequencing required for SNP discovery. In this study, a combination of reference-based and reference-free approaches were used to identify SNPs in Nordmann fir (Abies nordmanniana), a species previously lacking genomic sequence information. Using a combination of a genome assembly of the closely related Silver fir (Abies alba) species and a de novo assembly of low-copy regions of the Nordmann fir genome, we identified a high density of reliable SNPs. Reference-based approaches identified two million SNPs in common between the Silver fir genome and low-copy regions of Nordmann fir. A combination of one reference-free and two reference-based approaches identified 250 shared SNPs. A subset of 200 SNPs were used to genotype 342 individuals and thereby tested and validated in the context of identity analysis and/or clone identification. The tested SNPs successfully identified all ramets per clone and five mislabeled individuals via identity and genomic relatedness analysis. The identified SNPs will be used in ad hoc breeding of Nordmann fir in Denmark.

Unveiling dominant fungal pathogens associated with root rot of hybrid bermudagrass based on culture dependent and independent method.

Hybrid bermudagrass (Cynodon dactylon × C. transvaalensis) is widely used as turf in southern and transition zones of China. From June to September in 2022, an unknown disease was consistently observed on hybrid bermudagrass in different regions of Nanjing China, exhibiting distinct symptoms of leaf necrosis, severe root rot and circular or irregular necrotic patches with 20-300 cm in diameter. In this study, culture -independent and dependent methods were used to elucidate the dominant fungal pathogens associated with the disease. Basidiomycota and Marasmiellus were shown to be the dominant phyla (51.96%-70.60%) and genera (50.09%-69.84%) in the symptomatic samples. A total of 128 fungal strains were isolated from symptomatic root tissues, and 40 strains representing the largest proportion (31.25%), were identified as Marasmiellus mesosporus, based on the morphological characteristics, phylogenetic analysis of ITS and LSU rDNA region, and pathogenicity testing. Temperature sensitivity tests revealed that M. mesosporus grew well at high temperature (growth rate of 13.74 mm/d at 36 ℃). To our knowledge, this is the first report of M. mesosporus causing root rot disease on hybrid bermudagrass during hot summer months. The study will have important implications for the management of the disease.

Comparative genomics and transcriptome analysis reveals potential pathogenic mechanisms of Microdochium paspali on seashore paspalum.

The sparse leaf patch of seashore paspalum (Paspalum vaginatum Sw.) caused by Microdochium paspali seriously impacts the landscape value of turf and poses a challenge to the maintenance and management of golf courses. Little is known about the genome of M. paspali or the potential genes underlying pathogenicity. In this study, we present a high-quality genome assembly of M. paspali with 14 contigs using the Nanopore and Illumina platform. The M. paspali genome is roughly 37.32 Mb in size and contains 10,365 putative protein-coding genes. These encompass a total of 3,830 pathogen-host interactions (PHI) genes, 481 carbohydrate-active enzymes (CAZymes) coding genes, 105 effectors, and 50 secondary metabolite biosynthetic gene clusters (SMGCs) predicted to be associated with pathogenicity. Comparative genomic analysis suggests M. paspali has 672 species-specific genes (SSGs) compared to two previously sequenced non-pathogenic Microdochium species, including 24 species-specific gene clusters (SSGCs). Comparative transcriptomic analyses reveal that 739 PHIs, 198 CAZymes, 40 effectors, 21 SMGCs, 213 SSGs, and 4 SSGCs were significantly up-regulated during the process of infection. In conclusion, the study enriches the genomic resources of Microdochium species and provides a valuable resource to characterize the pathogenic mechanisms of M. paspali.

Genome resources and whole genome resequencing of Phytophthora rubi isolates from red raspberry.

Phytophthora rubi is a primary causal agent of Phytophthora root rot and wilting of raspberry (Rubus idaeus L.) worldwide. The disease is a major concern for raspberry growers in Canada and USA. To date, no information is available on genomic diversity of P. rubi population from raspberry in Canada. Using a PCR-free library prep with dual-indexing for an Illumina HiSEQX running a 2x150 bp configuration, we generated whole genome sequence data of P. rubi isolates (n = 25) recovered during 2018 to 2020 from nine fields, four locations and four cultivars of raspberry growing areas of British Columbia, Canada. The assembled genome of 24 isolates of P. rubi averaged 8,541 scaffolds, 309× coverage, and 65,960,000 bp. We exploited single nucleotide polymorphisms (SNPs) obtained from whole genome sequence data to analyze the genome structure and genetic diversity of the P. rubi isolates. Low heterozygosity among the 72% of pathogen isolates and standardized index of association revealed that those isolates were clonal. Principal component analysis, discriminant analysis of principal component, and phylogenetic tree revealed that P. rubi isolates clustered with the raspberry specific cultivars. This study provides novel resources and insight into genome structure, genetic diversity, and reproductive biology of P rubi isolated from red raspberry. The availability of the P. rubi genomes also provides valuable resources for future comparative genomic and evolutionary studies for oomycetes pathogens.

Screening for resistance alleles to Cry1 proteins through targeted sequencing in the native and invasive range of Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a highly polyphagous pest native to the tropical Americas that has recently spread to become a global super-pest threatening food and fiber production. Transgenic crops producing insecticidal Cry and Vip3Aa proteins from Bacillus thuringiensis (Bt) are used for control of this pest in its native range. The evolution of practical resistance represents the greatest threat to sustainability of this technology and its potential efficacy in the S. frugiperda invasive range. Monitoring for resistance is vital to management approaches delaying S. frugiperda resistance to Bt crops. DNA-based resistance screening provides higher sensitivity and cost-effectiveness than currently used bioassay-based monitoring. So far, practical S. frugiperda resistance to Bt corn-producing Cry1F has been genetically linked to mutations in the SfABCC2 gene, providing a model to develop and test monitoring tools. In this study, we performed targeted SfABCC2 sequencing followed by Sanger sequencing to confirm the detection of known and candidate resistance alleles to Cry1F corn in field-collected S. frugiperda from continental USA, Puerto Rico, Africa (Ghana, Togo, and South Africa), and Southeast Asia (Myanmar). Results confirm that the distribution of a previously characterized resistance allele (SfABCC2mut) is limited to Puerto Rico and identify 2 new candidate SfABCC2 alleles for resistance to Cry1F, one of them potentially spreading along the S. frugiperda migratory route in North America. No candidate resistance alleles were found in samples from the invasive S. frugiperda range. These results provide support for the potential use of targeted sequencing in Bt resistance monitoring programs.

Activity of the Succinate Dehydrogenase Inhibitor Fungicide Benzovindiflupyr Against Clarireedia spp.

Dollar spot (DS), caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa), is one of the most important diseases of turfgrasses worldwide. Benzovindiflupyr, a pyrazole carboxamide fungicide belonging to succinate dehydrogenase inhibitors, was recently registered for DS control. In this study, baseline sensitivity, toxicity, and control efficacy of benzovindiflupyr against Clarireedia spp. were evaluated. The frequency of sensitivities had a unimodal distribution (Kolmogorov-Smirnov, P > 0.10). The mean EC50 value was 1.109 ± 0.555 µg/ml, with individual values ranging from 0.160 to 2.548 µg/ml. Benzovindiflupyr increased the number of hyphal offshoots and cell membrane permeability and inhibited oxalic acid production. Positive cross-resistance was observed between benzovindiflupyr and boscalid, but not between benzovindiflupyr and thiophanate-methyl, propiconazole, or iprodione. Benzovindiflupyr showed high protective and curative control efficacies in vivo and in field applications. Both protective and curative control efficacies of benzovindiflupyr were significantly better than propiconazole, and equivalent to boscalid, over 2 years of field research. The results have important implications for managing DS and fungicide resistance problems in Clarireedia spp.

Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.

Using targeted sequencing and TaqMan approaches to detect acaricide (bifenthrin, bifenazate, and etoxazole) resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yards.

Tetranychus urticae (Koch) is an economically important pest of many agricultural commodities world-wide. Multiple acaricides, including bifenazate, bifenthrin, and extoxazole, are currently registered to control T. urticae. However, populations of T. urticae in many different growing regions have developed acaricide resistance through multiple mechanisms. Within T. urticae, single nucleotide polymorphisms (SNPs) have been documented in different genes which are associated with acaricide resistance phenotypes. The detection of these mutations through TaqMan qPCR has been suggested as a practical, quick, and reliable tool to inform agricultural producers of acaricide resistance phenotypes present within their fields and have potential utility for making appropriate acaricide application and integrated pest management decisions. Within this investigation we examined the use of a TaqMan qPCR-based approach to determine genotypes which have been previously associated with acaricide resistance in field-collected populations of T. urticae from peppermint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. The results suggest that a TaqMan qPCR approach accurately genotypes T. urticae populations for SNPs that have been linked to Bifenazate, Bifenthrin, and Etoxazole resistance. The results also demonstrated that different populations of mites in Washington and Idaho displayed varying frequencies of the examined SNPs. While we were able to detect the SNPs associated with the examined acaricides, the mutation G126S was not an appropriate or accurate indicator for bifenazate resistance.

First report of Phytophthora gonapodyides causing root rot on raspberry in Canada.

Raspberry (Rubus idaeus L.) is an economically important fruit crop in Canada and about 80% of red raspberries are cultivated in British Columbia. In 2018, foliar symptoms associated with root rot and wilting complex disease were observed in raspberry field of Fraser Valley areas of British Columbia. Plants were stunted with reduced numbers of primocanes. Chlorosis and necrosis on leaves and partial wilting of branches were observed. When plants were uprooted, necrosis and browning on roots were observed. Two isolates of oomycetes pathogen were isolated using baiting with rhododendron leaves and pear fruit as described in Sapkota et al. 2022. Using FastDNA Spin kit (MP Biomedical, Burlingame, CA), genomic DNA of pathogen isolates was extracted from mycelia cultured on 20% clarified V8 agar medium amended with 10 mg pimaricin, 250 mg ampicillin, 10 mg rifampicin (V8PAR) per liter following the manufacturer's standard protocol. Pathogens were identified using colony morphology on 20% clarified V8 PAR as well as internal transcribed spacer (ITS) sequencing with ITS1 primers (White et al. 1990) and multiplex targeted-sequencing with degenerate primers of three nuclear genes: heat shock protein90 (HSP90), elongation factor 1 alpha (EF1α) and beta tubulin (ßtub). BLAST searches of ITS sequences of isolates of this study (accession nos. OP180065, OP180066) in NCBI GenBank showed 98.5 to 99.6% identity with the ITS sequence of P. gonapodyides (accession nos. MN513238.1, MG753496.1). Multiplex targeted sequencing also identified both isolates as a P. gonapodyides (accession nos. SRR20227809, SRR20227807) when mapped with the reference sequences (accession nos. HSP90: KX251233.1, EF1α: KX251231.1, ß-tub: KX639710.1). Pathogenicity was confirmed by inoculating mycelial suspension of one isolate of P. gonapodyides on root of intact plants and mycelial plugs of two isolates on detached stems of the raspberry plants, 'Chemainus' in the greenhouse using methods described in Sapkota et al. 2022. Two experiments were conducted with three replicates in each test. Experiments were arranged using completely randomized design. In detached stem assays, distinct dark-lesion symptom appeared at 7 to 9 days after inoculation while uninoculated control stems remained asymptomatic. Intact plants showed wilting and foliar symptoms 15 days after inoculation and progressed higher at 4 to 5 weeks after inoculation. Root infection with dark brown to black color was observed when roots were assessed at 5 weeks after inoculation. The diseased root and crown tissues tested positive for Phytophthora in Agdia ImmunoStrip and P. gonapodyides was re-isolated and confirmed with multiplex-targeted sequencing. Phytophthora gonapodyides was previously reported from raspberry in Chile (Wilcox and Latorre 2002). To our best knowledge, this is the first report of P. gonapodyides infecting red raspberry in British Columbia, Canada. The detection of new Phytophthora species on raspberry may become a new potential problem to growers in addition to P. rubi, which is already a major cause of raspberry decline in the region.

A one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing dollar spot on turfgrass.

BACKGROUND: Dollar spot (DS) is one of the most destructive and economically important diseases of cool- and warm-season turfgrasses worldwide. A total of six species causing DS disease in the genus Clarireedia have been described, and four of them have been reported to be distributed countrywide in China. Identification of different species of Clarireedia is a prerequisite for the effective management of DS disease. RESULTS: Here we report a novel polymerase chain reaction (PCR)-based method for the detection and differentiation of the four species of Clarireedia associated with DS on turfgrass in China: C. jacksonii, C. paspali, C. monteithiana and C. hainanense. Species-specific genes were identified for each species by comparative genomics analysis. Four primer pairs were designed and mixed to amplify species-specific PCR fragments with differential sizes for the four species of Clarireedia in a single multiplex PCR assay. No PCR products were generated from the DNA templates of other common fungal pathogens associated with multiple turfgrass diseases. The multiplex PCR method developed can be used for the rapid and accurate detection and differentiation of the four species of Clarireedia from pure cultures as well as from infected turfgrass blades with DS symptoms. CONCLUSION: The study developed a one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing DS on turfgrass in China, which will have important implications for DS management in China and worldwide. © 2022 Society of Chemical Industry.

Genome Resources for Four Clarireedia Species Causing Dollar Spot on Diverse Turfgrasses.

Dollar spot (DS) is a destructive fungal disease impacting almost all warm- and cool-season turfgrasses worldwide. Multiple fungal species in the genus Clarireedia are causal agents of DS. Here, we present whole-genome assemblies of nine fungal isolates in the genus Clarireedia, including four species (C. paspali, C. hainanense, C. jacksonii, and C. monteithiana) causing DS on seashore paspalum (Paspalum vaginatum Sw.), creeping bentgrass (Agrostis stolonifera L.), and Kentucky bluegrass (Poa pratensis L.) in China. This work provides valuable baseline genomic data to support further research and management of DS pathogens on turfgrasses.

Phytophthora capsici, 100 Years Later: Research Mile Markers from 1922 to 2022.

In 1922, Phytophthora capsici was described by Leon Hatching Leonian as a new pathogen infecting pepper (Capsicum annuum), with disease symptoms of root rot, stem and fruit blight, seed rot, and plant wilting and death. Extensive research has been conducted on P. capsici over the last 100 years. This review succinctly describes the salient mile markers of research on P. capsici with current perspectives on the pathogen's distribution, economic importance, epidemiology, genetics and genomics, fungicide resistance, host susceptibility, pathogenicity mechanisms, and management.

High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing.

A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.

Strive or thrive: Trends in Phytophthora capsici gene expression in partially resistant pepper.

Partial resistance in plants generally exerts a low selective pressure on pathogens, and thus ensuring their durability in agrosystems. However, little is known about the effect of partial resistance on the molecular mechanisms of pathogenicity, a knowledge that could advance plant breeding for sustainable plant health. Here we investigate the gene expression of Phytophthora capsici during infection of pepper (Capsicum annuum L.), where only partial genetic resistance is reported, using Illumina RNA-seq. Comparison of transcriptomes of P. capsici infecting susceptible and partially resistant peppers identified a small number of genes that redirected its own resources into lipid biosynthesis to subsist on partially resistant plants. The adapted and non-adapted isolates of P. capsici differed in expression of genes involved in nucleic acid synthesis and transporters. Transient ectopic expression of the RxLR effector genes CUST_2407 and CUST_16519 in pepper lines differing in resistance levels revealed specific host-isolate interactions that either triggered local necrotic lesions (hypersensitive response or HR) or elicited leave abscission (extreme resistance or ER), preventing the spread of the pathogen to healthy tissue. Although these effectors did not unequivocally explain the quantitative host resistance, our findings highlight the importance of plant genes limiting nutrient resources to select pepper cultivars with sustainable resistance to P. capsici.

Development and application of multiplex targeted-sequencing approaches to identify Phytophthora species associated with root rot and wilting complex of red raspberry.

Phytophthora species are primary causal agents of raspberry root rot and wilting complex (RRWC), a disease complex that is of major concern to raspberry producers worldwide. Accurate identification of the causal agents is a first step for effective disease management. Advancements in molecular diagnostics can facilitate the detection of multiple pathogen species associated with this disease complex. We developed multiplex targeted-sequencing methods using degenerate primers for heat shock protein 90, elongation factor 1α and ß-tubulin genes to identify Phytophthora species causing RRWC. One hundred and twenty-eight isolates recovered during 2018 to 2020 from diverse fields in major raspberry growing areas of British Columbia (BC) were sequenced and identified by comparing with known reference sequences of 142 Phytophthora species, 111 Pythium species, and nine Phytopythium species in the NCBI database. This multiplex targeted-sequencing method was highly specific and identified two species of Phytophthora associated with RRWC. These were P. rubi (85% of isolates) and P. gonapodyides (15% of isolates). Phytophthora rubi was predominantly isolated from the cultivars 'Chemainus' (51%), 'Rudi' (27%) and 'Meeker' (15%), whereas P. gonapodyides was predominately isolated from the moderately resistant cultivar 'Cascade Bounty'. Pathogenicity studies on intact plants and detached leaves confirmed that P. rubi and P. gonapodyides can cause symptoms of RRWC on raspberry, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. gonapodyides as a causal agent of RRWC on raspberry in BC. This study provides novel insights into the identification and species composition of Phytophthora associated with RRWC in raspberry production systems.

Distribution and Prevalence of Plant-Parasitic Nematodes of Turfgrass at Golf Courses in China.

We sampled 127 turfgrass soil samples from 33 golf courses in NC, EC, and SC for plant-parasitic nematodes (PPNs). PPNs were extracted from soil samples using the shallow dish method and were identified at the genus or species levels with a combination of morphological and molecular methods. The results revealed 41 species of nematode belonging to 20 genera and 10 families. Nine genera are new records of PPNs associated with turfgrass in China. The PPNs show strong geographical distributions. Of the 20 genera, Helicotylenchus, Paratrichodorus, Hoplolaimus, Meloidogyne, Hemicriconemoides, and Mesocriconema showed higher infestation and frequency, and most of these genera had numbers in soil samples above established damage thresholds. Four golf courses had soil samples with PPNs > 30%, indicating the potential for nematode damage. The biodiversity indices H', SR, J', λ, and H2 showed significant differences among different regions and turfgrass species; H', SR, J', and H2 were significantly higher in EC than in NC and SC, while λ was lowest in EC. Creeping bentgrass had the highest H', SR, J', and H2 and the lowest λ in comparison with seashore paspalum and hybrid bermudagrass. These findings provide baseline information on the occurrence of turfgrass-associated PPNs in China, and have important implications for the effective management of PPNs causing damage on turfgrass.

Multiplex Polymerase Chain Reaction Reveals Unique Trends in Pathogen and Parasitoid Infestations of Alfalfa Leafcutting Brood Cells.

The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. M. rotundata is a solitary cavity nesting bee that forms brood nests where its larvae can develop. During the developmental stages of growth, brood can be preyed upon by multiple different fungal pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollination services provided by M. rotundata. Reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. The presence of multiple Ascosphaera species in a single brood cell was observed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species, including multiple predators and pathogenetic species not previously documented in association with M. rotundata brood cells.

Clarireedia hainanense: A New Species Is Associated with Dollar Spot of Turfgrass in Hainan, China.

The genus Clarireedia contains multiple species causing dollar spot (DS) on turfgrass worldwide. In November 2020, 119 Clarireedia isolates were obtained from symptomatic seashore paspalum at golf courses in Hainan province and identified to species level based on partial sequence of the internal transcribed spacer (ITS) region. A total of 45 and 22 isolates were identified as C. paspali and C. monteithiana, respectively; the remaining 52 isolates defined a new clade. Isolates from this clade were further selected for phylogenetic, morphological, and biological analyses. Maximum likelihood and Bayesian methods were implemented to obtain phylogenetic trees for partial sequences of the ITS, EF-1α, and McM7 genes. The selected isolates consistently fell into a distinct, well-supported clade within Clarireedia. Morphological and biological characteristics were observed among the different species in Clarireedia. Altogether, this study described a new species, Clarireedia hainanense, which has widespread distribution in Hainan, China. These findings may have important implications for the management of DS disease.