The Impatiens Downy Mildew Epidemic in the United States Is Caused by New, Introgressed Lineages of Plasmopara destructor with Prominent Genotypic Diversity and High Evolutionary Potential.

Impatiens downy mildew (IDM) caused by Plasmopara destructor is currently the primary constraint on the production and use of impatiens (Impatiens walleriana) as bedding plants worldwide. Downy mildew has been documented since the 1880s from wild-grown Impatiens spp. but epidemic outbreaks of the disease affecting the commercially grown, ornamental I. walleriana were only reported for the first time in 2003 in the United Kingdom and in 2004 in the United States. Here, we assess the genetic diversity, level of differentiation, and population structure from 623 samples associated with current and preepidemic IDM outbreaks, by genotyping the samples with simple sequence repeat markers. P. destructor population structure following the emergence of IDM in the United States is subdivided into four genetic lineages characterized by high genetic diversity, mixed reproduction mode, inbreeding, and an excess of heterozygosity. P. destructor genotypes are significantly differentiated from preepidemic IDM samples from hosts other than I. walleriana but no geographical or temporal subdivision is evident. P. destructor samples from different Impatiens spp. show significant but very low levels of differentiation in the analysis of molecular variance test that did not hold in discriminant analysis of principal components analyses. The same was observed between samples of P. destructor and P. velutina recovered from I. walleriana. The finding of shared genotypes in samples from different countries and lack of differentiation among U.S. and Costa Rican samples indicate the occurrence of international movement of the pathogen. Our study provides the first high-resolution analysis of the diversity of P. destructor populations and the IDM epidemic that may be instrumental for disease management and breeding efforts.

Mitochondrial Loci Enable Specific Quantitative Real-Time PCR Detection of the Pathogen Causing Contemporary Impatiens Downy Mildew Epidemics.

Impatiens downy mildew (IDM) disease is a primary constraint on the production of Impatiens walleriana, a popular and economically important floriculture plant. IDM is caused by the biotrophic. oomycete Plasmopara destructor that emerged as a pathogen of I. walleriana in the 2000s. To enable P. destructor detection and quantification, a hydrolysis-probe-based quantitative PCR diagnostic assay was developed based on unique orientation and order of the mitochondrial cytochrome c oxidase subunit1 (cox1) and ATP synthase subunit alpha (atp1) genes in the genus Plasmopara. Nucleotide sequences and analysis of the cox1/atp1 region distinguished P. destructor and its sister-species P. obducens, consistent with prior phylogenetic analyses using cox2 and rDNA markers. Specificity for P. destructor was incorporated into a hydrolysis probe targeting the cox1 gene and flanking primers that amplified across the cox1/atp1 intergenic region. The limit of detection was 0.5 fg/µl of P. destructor DNA (∼100 plasmid copies/µl), with amplification efficiency = 0.95. The assay was validated against a panel of target and nontarget oomycetes, which showed that the primers were specific for Plasmopara spp., while the probe was specific for P. destructor infecting both I. walleriana and I. balsamina. Testing of Impatiens tissue collected from 23 locations across 13 states indicated all samples with IDM symptoms tested positive for P. destructor. Asymptomatic plants from two locations also tested positive for P. destructor.

First report: Co-infection of Sarcococca hookeriana (sweetbox) by Coccinonectria pachysandricola and Calonectria pseudonaviculata causes a foliar disease of sweetbox in Pennsylvania.

Sweetbox (Sarcococca hookeriana) are high value ornamental shrubs susceptible to disease caused by Calonectria pseudonaviculata (Cps) and Coccinonectria pachysandricola (Cpa) (Malapi-Wight et al. 2016; Salgado-Salazar et al. 2019). In July 2018, 18-month old sweetbox with leaf spots and defoliation were observed in a residential landscape in Lancaster County, Pennsylvania. Small tan leaf spots grew to cover half of the leaf, developing a concentric banding with dark brown rings and a yellow halo (Sup. Doc. 1: Sup. Fig. 1). The symptoms agreed with those of Cpa disease of sweetbox reported from Washington D.C. (Salgado-Salazar et al. 2019). Diseased plants were located ~1.5 m from Buxus sempervirens with boxwood blight. Morphological and genetic characterization of isolated fungi and pathogenicity tests followed Salgado-Salazar et al. (2019) (Sup. Doc. 2). White to salmon pink spore masses developed on the abaxial leaf surface after humid chamber incubation. Two distinct fungal cultures were recovered (JAC 18-61, JAC 18-79) on potato dextrose agar (Fisher Scientific, Pittsburg, PA). JAC 18-61 presented cultural and morphological characteristics as described for Cps (Crous et al. 2002). JAC 18-79 produced flat, filamentous, light salmon colonies with tan centers and white filiform borders containing pale pink sporodochia, verticillate and simple conidiophores (x̄: 61.8 ± 20.12 µm, N = 20) with lateral, cylindrical phialides (x̄ = 18.1 ± 5.83 x 2.4 ± 0.7 µm, N = 20), and ellipsoid, hyaline conidia without septa (x̄ = 15.2 ± 1.9 x 3.3 ± 0.7 µm, N = 20). Sexual structures and chlamydospores were not observed. The characteristics of JAC 18-79 agree with those reported for Cpa (Salgado-Salazar et al. 2019). Bidirectional sequencing of the ITS, beta-TUB, and RPB1 and RPB2 regions was performed as described (Salgado-Salazar et al. 2019). BLASTn comparisons against NCBI GenBank revealed JAC 18-61 sequences (MT318150 and MT328399) shared 100% identity with Cps sequences (JX535321 and JX535307 from isolate CB002). Sequences from JAC 18-79 (MT318151, MT341237 to MT341239) were 100% identical to Cpa sequences (MH892596, MH936775, MH936703 from isolate JAC 16-20 and JF832909, isolate CBS 128674). The genome of JAC 18-79 was sequenced and yielded an assembly of 26.3 Mb (204 contigs > 1000 bases, N50 = 264.3 kb, 92x coverage, JABAHV0000000000) that contained the MAT1-2 mating-type idiomorph and shared 98.9% similarity with Cpa BPI910731. Isolate JAC 18-61 (Cps) caused lesions on wounded and unwounded sweetbox and boxwood leaves (Sup. Table 1). In general, JAC 18-79 (Cpa) infected only wounded leaves of both hosts; however, in one trial, one unwounded sweetbox and two unwounded boxwood plants developed lesions, possibly due to the presence of natural wounds. Control plants did not develop symptoms. These results diverge to some degree from previous reports of Cpa infecting unwounded sweetbox and not infecting wounded boxwood (Salgado-Salazar et al. 2019). These results indicate that virulence variation among Cpa isolates might occur. Plating of symptomatic tissue and examination of spores fulfilled Koch's postulates for both pathogens. To our knowledge, this is the first report of Cpa blight on sweetbox in Pennsylvania, and the second U.S. report of the disease. This is also the first report of co-infection of Cpa and Cps on diseased sweetbox foliage. Given the capacity of Cpa to infect both sweetbox and boxwood, inspection for Cpa on both hosts is advisable.

Sporangiospore Viability and Oospore Production in the Spinach Downy Mildew Pathogen, Peronospora effusa.

Downy mildew of spinach, caused by the obligate pathogen Peronospora effusa, remains the most important constraint in the major spinach production areas in the United States. This disease can potentially be initiated by asexual sporangiospores via "green bridges", sexually derived oospores from seed or soil, or dormant mycelium. However, the relative importance of the various types of primary inoculum is not well known. The ability of P. effusa sporangiospores to withstand abiotic stress, such as desiccation, and remain viable during short- and long-distance dispersal and the ability of oospores to germinate and infect seedlings remain unclear. Thus, the primary objectives of this research were to evaluate the impact of desiccation on sporangiospore survival and infection efficiency and examine occurrence, production, and germination of oospores. Results indicate that desiccation significantly reduces sporangiospore viability as well as infection potential. Leaf wetness duration of 4 h was needed for disease establishment by spinach downy mildew sporangiospores. Oospores were observed in leaves of numerous commercial spinach cultivars grown in California in 2018 and Arizona in 2019. Frequency of occurrence varied between the two states-years. The presence of opposite mating types in spinach production areas in the United States was demonstrated by pairing isolates in controlled crosses and producing oospores on detached leaves as well as intact plants. Information from the study of variables that affect sporangiospore viability and oospore production will help in improving our understanding of the epidemiology of this important pathogen, which has implications for management of spinach downy mildew.

One Clonal Lineage of Calonectria pseudonaviculata Is Primarily Responsible for the Boxwood Blight Epidemic in the United States.

Boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae is destroying cultivated and native boxwood worldwide, with profound negative economic impacts on the horticulture industry. First documented in the United States in 2011, the disease has now occurred in 30 states. Previous research showed that global C. pseudonaviculata populations prior to 2014 had a clonal structure, and only the MAT1-2 idiomorph was observed. In this study, we examined C. pseudonaviculata genetic diversity and population structure in the United States after 2014, following the expansion of the disease across the country over the past 5 years. Two hundred eighteen isolates from 21 states were genotyped by sequencing 11 simple sequence repeat (SSR) loci and by MAT1 idiomorph typing. All isolates presented C. pseudonaviculata-specific alleles, indicating that C. henricotiae is still absent in the U.S. states sampled. The presence of only the MAT1-2 idiomorph and gametic linkage disequilibrium suggests the prevalence of asexual reproduction. The contemporary C. pseudonaviculata population is characterized by a clonal structure and composed of 13 multilocus genotypes (SSR-MLGs) unevenly distributed across the United States. These SSR-MLGs grouped into two clonal lineages (CLs). The predominant lineage CL2 (93% of isolates) is the primary contributor to U.S. disease expansion. The contemporary U.S. C. pseudonaviculata population is not geographically subdivided and not genetically differentiated from the U.S. population prior to 2014, but is significantly differentiated from the main European population, which is largely composed of CL1. Our findings provide insights into the boxwood blight epidemic that are critical for disease management and breeding of resistant boxwood cultivars.

Wheat blast: from its origins in South America to its emergence as a global threat.

Wheat blast was first reported in Brazil in 1985. It spread rapidly across the wheat cropping areas of Brazil to become the most important biotic constraint on wheat production in the region. The alarming appearance of wheat blast in Bangladesh in 2016 greatly increased the urgency to understand this disease, including its causes and consequences. Here, we summarize the current state of knowledge of wheat blast and aim to identify the most important gaps in our understanding of the disease. We also propose a research agenda that aims to improve the management of wheat blast and limit its threat to global wheat production.

Wheat Blast: Past, Present, and Future.

The devastating wheat blast disease first emerged in Brazil in 1985. The disease was restricted to South America until 2016, when a series of grain imports from Brazil led to a wheat blast outbreak in Bangladesh. Wheat blast is caused by Pyricularia graminis-tritici ( Pygt), a species genetically distinct from the Pyricularia oryzae species that causes rice blast. Pygt has high genetic and phenotypic diversity and a broad host range that enables it to move back and forth between wheat and other grass hosts. Recombination is thought to occur mainly on the other grass hosts, giving rise to the highly diverse Pygt population observed in wheat fields. This review brings together past and current knowledge about the history, etiology, epidemiology, physiology, and genetics of wheat blast and discusses the future need for integrated management strategies. The most urgent current need is to strengthen quarantine and biosafety regulations to avoid additional spread of the pathogen to disease-free countries. International breeding efforts will be needed to develop wheat varieties with more durable resistance.

Population Genetic Structure of Rhizoctonia oryzae-sativae from Rice in Latin America and Its Adaptive Potential to Emerge as a Pathogen on Urochloa Pastures.

The fungus Rhizoctonia oryzae-sativae is an important pathogen that causes the aggregated sheath spot disease on rice. In this study, we investigated the genetic structure of rice-adapted populations of R. oryzae-sativae sampled from traditional rice-cropping areas from the Paraíba Valley, São Paulo, Brazil, and from Meta, in the Colombian Llanos, in South America. We used five microsatellite loci to measure population differentiation and infer the pathogen's reproductive system. Gene flow was detected among the three populations of R. oryzae-sativae from lowland rice in Brazil and Colombia. In contrast, a lack of gene flow was observed between the lowland and the upland rice populations of the pathogen. Evidence of sexual reproduction including low clonality, Hardy-Weinberg equilibrium within loci and gametic equilibrium between loci, indicated the predominance of a mixed reproductive system in all populations. In addition, we assessed the adaptive potential of the Brazilian populations of R. oryzae-sativae to emerge as a pathogen to Urochloa spp. (signalgrass) based on greenhouse aggressiveness assays. The Brazilian populations of R. oryzae-sativae were probably only incipiently adapted as a pathogen to Urochloa spp. Comparison between RST and QST showed the predominance of diversifying selection in the divergence between the two populations of R. oryzae-sativae from Brazil.

Web blight (Thanatephorus cucumeris): a new disease on leaves of okra plants / Mancha areolada (Thanatephorus cucumeris): nova doença das folhas do quiabeiro

In an experiment on organic production of okra (Abelmoschus esculentus (L.) Moench) that was carried out from September 2013 to January 2014, in Manaus, Amazonas state, Brazil, we observed large chlorotic, necrotic, helical, discontinuous, dark or light-brown lesions with partial detachment of the injured area on the adaxial surface of leaves located in the median and basal portions of the plants. A whitish mycelium mantle covers the lesions on the leaves at the abaxial surface at high moisture conditions. Using morphological characteristics, Koch's postulates, and phylogenetic analyses of the ITS-5.8S rDNA region, we identified that the fungus causing the lesions on the okra leaves was Thanatephorus cucumeris (Frank) Donk (asexual stage of Rhizoctonia solani Kuhn of the anastomosis group AG-1 ID). This is the first report of T. cucumeris causing web blight on okra in Brazil, and probably in the world. So far, T. cucumeris was described on okra only on post-harvest pods rotting and seedlings' damping off.(AU)

Em um experimento sobre a produção orgânica do quiabeiro (Abelmoschus esculentus (L.) Moench), que foi instalado em Manaus, Amazonas, Brasil, no período de setembro de 2013 a janeiro de 2014, observou-se, na face adaxial do limbo foliar das folhas medianas e baixeiras, a ocorrência de lesões cloróticas e necróticas grandes, helicoidais, de coloração marrom escuro ou marrom claro e descontínuas, com desprendimento parcial da área lesionada. Na face abaxial, sobre as manchas, em condições de alta umidade, constatou-se a presença de um manto micelial esbranquiçado do patógeno, facilmente visível, recobrindo a área colonizada. Por meio da análise de características morfológicas, postulados de Koch e análise filogenética da região ITS-5.8S do rDNA do fungo isolado, identificou-se Thanatephorus cucumeris (Frank) Donk (fase assexuada Rhizoctonia solani Kuhn grupo de anastomose AG-1 ID) como o agente causal da doença. Este é o primeiro relato de T. cucumeris causando mancha foliar em quiabeiro no Brasil e, provavelmente, no mundo. Até então, sua ocorrência em quiabeiro estava restrita à podridão pós-colheita em frutos e tombamento de mudas.(AU)

Emergence of wheat blast in Bangladesh was caused by a South American lineage of Magnaporthe oryzae.

BACKGROUND: In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16 % of the cultivated wheat area in Bangladesh, with yield losses reaching up to 100 %. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields. RESULTS: Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae. CONCLUSION: Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.

Resistance to QoI Fungicides Is Widespread in Brazilian Populations of the Wheat Blast Pathogen Magnaporthe oryzae.

Wheat blast, caused by Magnaporthe oryzae, is an important disease across central and southern Brazil. Control has relied mainly on strobilurin fungicides (quinone-outside inhibitors [QoIs]). Here, we report the widespread distribution of QoI resistance in M. oryzae populations sampled from wheat fields and poaceous hosts across central and southern Brazil and the evolution of the cytochrome b (cyt b) gene. Sequence analysis of the cyt b gene distinguished nine haplotypes, with four haplotypes carrying the G143A mutation associated with QoI resistance and two haplotypes shared between isolates sampled from wheat and other poaceous hosts. The frequency of the G143A mutation in the wheat-infecting population increased from 36% in 2005 to 90% in 2012. The G143A mutation was found in many different nuclear genetic backgrounds of M. oryzae. Our findings indicate an urgent need to reexamine the use of strobilurins to manage fungal wheat diseases in Brazil.

Population structure and pathotype diversity of the wheat blast pathogen Magnaporthe oryzae 25 years after its emergence in Brazil.

Since its first report in Brazil in 1985, wheat blast, caused by Magnaporthe oryzae (anamorph: Pyricularia oryzae), has become increasingly important in South America, where the disease is still spreading. We used 11 microsatellite loci to elucidate the population structure of the wheat blast pathogen in wheat fields in central-western, southeastern, and southern Brazil. No subdivision was found among the wheat-infecting populations, consistent with high levels of gene flow across a large spatial scale. Although the clonal fraction was relatively high and the two mating type idiomorphs (MAT1-1 and MAT1-2) were not at similar frequencies, the clone-corrected populations from Distrito Federal and Goiás, Minas Triangle, and São Paulo were in gametic equilibrium. Based on these findings, we propose that populations of the wheat blast pathogen exhibit a mixed reproductive system in which sexual reproduction is followed by the local dispersal of clones. Seedling virulence assays with local wheat cultivars differentiated 14 pathotypes in the current population. Detached head virulence assays differentiated eight virulence groups on the same wheat cultivars. There was no correlation between seedling and head reactions.