Development and evaluation of real-time quantitative PCR assays for detection of Phellinus noxius causing brown root rot disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood decay fungi, or host plant tissues. This study developed SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, five other Phellinus species, and 22 non-Phellinus wood decay fungal species. While all three primer pairs could detect as little as 100 fg (about 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff Cq values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

Investigation of Asymptomatic Infection of Phellinus noxius in Herbaceous Plants.

The white-rot fungus Phellinus noxius is known to cause brown root rot disease (BRRD) in woody trees and shrubs. To understand the pathogenicity of P. noxius in herbaceous plants, we investigated 23 herbaceous weed and turfgrass species in 32 BRRD-infested sites in Taiwan and/or tested them by artificial inoculation. In the field survey, P. noxius was isolated from seven symptomless herbaceous species (i.e., Typhonium blumei, Paspalum conjugatum, Paspalum distichum, Oplismenus compositus, Bidens pilosa, Digitaria ciliaris, and Zoysia matrella). Potted plant inoculation assays suggested that P. noxius is able to infect Artemisia princeps, O. compositus, and Z. matrella but not Axonopus compressus, Eremochloa ophiuroides, Ophiopogon japonicus, or Cynodon dactylon. A. princeps plants wilted within 2 weeks postinoculation, but inoculated O. compositus and Z. matrella were asymptomatic, and P. noxius could be isolated from only inoculated sites. The colonization of P. noxius in the cortex and vascular cylinder of roots was visualized by paraffin sectioning and trypan blue staining of juvenile seedlings grown on water agar. To evaluate the effect of replantation for the remediation of BRRD-infested sites, P. noxius-inoculated wood strips were buried in soil with or without vegetation. After 4 weeks, P. noxius could be detected only in the bare soil group. For the control of BRRD, the herbaceous hosts should be removed around the diseased trees/stumps and non-host turfgrasses (e.g., A. compressus, E. ophiuroides, O. japonicus, or C. dactylon) planted to accelerate the degradation of P. noxius.

Comparative genomic and transcriptomic analyses of trans-kingdom pathogen Fusarium solani species complex reveal degrees of compartmentalization.

BACKGROUND: The Fusarium solani species complex (FSSC) comprises fungal pathogens responsible for mortality in a diverse range of animals and plants, but their genome diversity and transcriptome responses in animal pathogenicity remain to be elucidated. We sequenced, assembled and annotated six chromosome-level FSSC clade 3 genomes of aquatic animal and plant host origins. We established a pathosystem and investigated the expression data of F. falciforme and F. keratoplasticum in Chinese softshell turtle (Pelodiscus sinensis) host. RESULTS: Comparative analyses between the FSSC genomes revealed a spectrum of conservation patterns in chromosomes categorised into three compartments: core, fast-core (FC), and lineage-specific (LS). LS chromosomes contribute to variations in genomes size, with up to 42.2% of variations between F. vanettenii strains. Each chromosome compartment varied in structural architectures, with FC and LS chromosomes contain higher proportions of repetitive elements with genes enriched in functions related to pathogenicity and niche expansion. We identified differences in both selection in the coding sequences and DNA methylation levels between genome features and chromosome compartments which suggest a multi-speed evolution that can be traced back to the last common ancestor of Fusarium. We further demonstrated that F. falciforme and F. keratoplasticum are opportunistic pathogens by inoculating P. sinensis eggs and identified differentially expressed genes also associated with plant pathogenicity. These included the most upregulated genes encoding the CFEM (Common in Fungal Extracellular Membrane) domain. CONCLUSIONS: The high-quality genome assemblies provided new insights into the evolution of FSSC chromosomes, which also serve as a resource for studies of fungal genome evolution and pathogenesis. This study also establishes an animal model for fungal pathogens of trans-kingdom hosts.

Analysis of the Pathogenicity and Phylogeny of Colletotrichum Species Associated with Brown Blight of Tea (Camellia sinensis) in Taiwan.

Brown blight, a destructive foliar disease of tea, has become a highly limiting factor for tea cultivation in Taiwan. To understand the population composition of the causal agents (Colletotrichum spp.), the fungal diversity in the main tea-growing regions all over Taiwan was surveyed from 2017 to 2019. A collection of 139 Colletotrichum isolates was obtained from 14 tea cultivars in 86 tea plantations. Phylogenic analysis using the ribosomal internal transcribed spacer, glutamine synthetase gene, Apn2-Mat1-2 intergenic spacer, ß-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase genes together with morphological characterization revealed three species associated with brown blight of tea; namely, Colletotrichum camelliae (95.6% of all isolates), C. fructicola (3.7%), and C. aenigma (0.7%). This is the first report of C. aenigma in Taiwan. The optimal growth temperatures were 25°C for C. camelliae and 25 and 30°C for C. fructicola and C. aenigma. Although C. fructicola and C. aenigma were more adapted to high temperature, C. camelliae was the most pathogenic across different temperatures. Regardless of whether spore suspensions or mycelial discs were used, significantly larger lesions and higher disease incidences were observed for wounded than for nonwounded inoculation and for the third and fourth leaves than for the fifth leaves. Wounded inoculation of detached third and fourth tea leaves with mycelial discs was found to be a reliable and efficient method for assessing the pathogenicity of Colletotrichum spp. within 4 days. Preventive application of fungicides or biocontrol agents immediately after tea pruning and at a young leaf stage would help control the disease.

Translocation of Fungicides and Their Efficacy in Controlling Phellinus noxius, the Cause of Brown Root Rot Disease.

Brown root rot disease (BRRD), caused by Phellinus noxius, is an important tree disease in tropical and subtropical areas. To improve chemical control of BRRD and deter emergence of fungicide resistance in P. noxius, this study investigated control efficacies and systemic activities of fungicides with different modes of action. Fourteen fungicides with 11 different modes of action were tested for inhibitory effects in vitro on 39 P. noxius isolates from Taiwan, Hong Kong, Malaysia, Australia, and Pacific Islands. Cyproconazole, epoxiconazole, and tebuconazole (Fungicide Resistance Action Committee [FRAC] 3, target-site G1) inhibited colony growth of P. noxius by 99.9 to 100% at 10 ppm and 97.7 to 99.8% at 1 ppm. The other effective fungicide was cyprodinil + fludioxonil (FRAC 9 + 12, target-site D1 + E2), which showed growth inhibition of 96.9% at 10 ppm and 88.6% at 1 ppm. Acropetal translocation of six selected fungicides was evaluated in bishop wood (Bischofia javanica) seedlings by immersion of the root tips in each fungicide at 100 ppm, followed by liquid or gas chromatography tandem mass spectrometry analyses of consecutive segments of root, stem, and leaf tissues at 7 and 21 days posttreatment. Bidirectional translocation of the fungicides was also evaluated by stem injection of fungicide stock solutions. Cyproconazole and tebuconazole were the most readily absorbed by roots and efficiently transported acropetally. Greenhouse experiments suggested that cyproconazole, tebuconazole, and epoxiconazole have a slightly higher potential for controlling BRRD than mepronil, prochloraz, and cyprodinil + fludioxonil. Because all tested fungicides lacked basipetal translocation, soil drenching should be considered instead of trunk injection for their use in BRRD control.

The brown root rot fungus Phellinus noxius affects microbial communities in different root-associated niches of Ficus trees.

Brown root rot (BRR) caused by Phellinus noxius is a destructive tree disease in tropical and subtropical areas. To understand how BRR affects the composition of the plant rhizoplane-enriched microbiota, the microbiomes within five root-associated compartments (i.e., bulk soil, old/young root rhizosphere soil, old/young root tissue) of Ficus trees naturally infected by P. noxius were investigated. The level of P. noxius infection was determined by quantitative PCR. Illumina sequencing of the internal transcribed spacer and 16S rRNA revealed that P. noxius infection caused a significant reduction in fungal diversity in the bulk soil, the old root rhizosphere soil, and the old root tissue. Interestingly, Cosmospora was the only fungal genus positively correlated with P. noxius. The abundance and composition of dominant bacterial taxa such as Actinomadura, Bacillus, Rhodoplanes, and Streptomyces differed between BRR-diseased and healthy samples. Furthermore, 838 isolates belonging to 26 fungal and 35 bacterial genera were isolated and tested for interactions with P. noxius. Antagonistic activities were observed for isolates of Bacillus, Pseudomonas, Aspergillus, Penicillium, and Trichoderma. Cellophane overlay and cellulose/lignin utilization assays suggested that Cosmospora could tolerate the secretions of P. noxius and that the degradation of lignin by P. noxius may create suitable conditions for Cosmorpora growth.

Surveillance of Rice Blast Resistance Effectiveness and Emerging Virulent Isolates in Taiwan.

Rice blast caused by Magnaporthe oryzae is a dangerous threat to rice production and food security worldwide. Breeding and proper deployment of resistant varieties are effective and environmentally friendly strategies to manage this notorious disease. However, a highly dynamic and quickly evolved rice blast pathogen population in the field has made disease control with resistance germplasms more challenging. Therefore, continued monitoring of pathogen dynamics and application of effective resistance varieties are critical tasks to prolong or sustain field resistance. Here, we report a team project that involved evaluation of rice blast resistance genes and surveillance of M. oryzae field populations in Taiwan. A set of International Rice Research Institute-bred blast-resistant lines (IRBLs) carrying single blast resistance genes was utilized to monitor the field effectiveness of rice blast resistance. Resistance genes such as Ptr (formerly Pita2) and Pi9 exhibited the best and most durable resistance against the rice blast fungus population in Taiwan. Interestingly, line IRBLb-B harboring the Pib gene with good field protection has recently shown susceptible lesions in some locations. To dissect the genotypic features of virulent isolates against the Pib resistance gene, M. oryzae isolates were collected and analyzed. Screening of the AvrPib locus revealed that the majority of field isolates still maintained the wild-type AvrPib status but eight virulent genotypes were found. Pot3 insertion appeared to be a major way to disrupt the AvrPib avirulence function. Interestingly, a novel AvrPib double-allele genotype among virulent isolates was first identified. Pot2 repetitive element-based polymerase chain reaction (rep-PCR) fingerprinting analysis indicated that mutation events may occur independently among different lineages in different geographic locations of Taiwan. This study provides our surveillance experience of rice blast disease and serves as the foundation to sustain rice production.

Cryptic Diversity, Molecular Systematics, and Pathogenicity of Genus Pestalotiopsis and Allied Genera Causing Gray Blight Disease of Tea in Taiwan, With a Description of a New Pseudopestalotiopsis Species.

Camellia sinensis (L.) O. Kuntze, commonly known as tea, is widely cultivated around the world in tropical and subtropical areas. Tea is mainly manufactured using young shoots of tea plants. Therefore, it is essential to control foliar diseases. Gray blight disease is caused by pestalotiopsis-like taxa and is known as one of the most destructive tea diseases. Although several studies have provided the groundwork for the fungal diseases associated with C. sinensis in Taiwan, gray blight disease has not been characterized based on diversity, molecular systematics, or pathogenicity. The goal of this study was to identify and characterize the causative agents of tea gray blight disease. A total of 98 pestalotiopsis-like isolates associated with symptomatic leaves of C. sinensis from major tea fields in Taiwan were investigated. Based on phylogenies of single and concatenated DNA sequences (internal transcribed spacer, ß-tubulin, translation elongation factor 1-α) together with morphology, we resolved most of the pestalotiopsis-like species in this study. The study revealed seven well-classified taxa and seven tentative clades in three genera: Pestalotiopsis, Pseudopestalotiopsis, and Neopestalotiopsis. One novel species, Pseudopestalotiopsis annellata, was introduced. Five new records, Pseudopestalotiopsis chinensis, Pseudopestalotiopsis camelliae-sinensis, Pestalotiopsis camelliae, Pestalotiopsis yanglingensis, and Pestalotiopsis trachicarpicola, were introduced for the first time in Taiwan. Pseudopestalotiopsis chinensis was the taxon most frequently isolated from C. sinensis in this study. Furthermore, results of pathogenicity assessments exhibited that, with wound inoculation, all assayed isolates in this study were pathogenic on tea leaves. Pseudopestalotiopsis chinensis and Pseudopestalotiopsis camelliae-sinensis were identified as the major pathogens associated with gray blight disease of tea in Taiwan. To our knowledge, this is the first study of the diversity, pathogenicity, and characterization of pestalotiopsis-like fungi causing tea gray blight disease in Taiwan.

Stemphylium Leaf Blight of Welsh Onion (Allium fistulosum): An Emerging Disease in Sanxing, Taiwan.

Welsh onion (Allium fistulosum L.) is one of the main and oldest vegetable crops grown in Taiwan. A severe epidemic of leaf blight in Welsh onion caused by a Stemphylium-like pathogen was found in Sanxing, Taiwan, from 2018 to 2020. However, correct species identification, biology, and control of Stemphylium leaf blight (SLB) of Welsh onion are not well-established. Therefore, the main objective of this study was to investigate the causal agent of SLB in Sanxing and evaluate the in vitro sensitivity of Stemphylium-like pathogen to commonly used fungicides. A phylogenetic analysis based on combining the internal transcribed spacer (ITS) region and glyceraldedyhe-3-phosphate dehydrogenase (gapdh) and calmodulin (cmdA) gene sequences together with morphological features identified that S. vesicarium is associated with SLB in Sanxing. When inoculated onto Welsh onion leaves, the isolates caused symptoms identical to those observed in the field; therefore, S. vesicarium was reisolated and Koch's postulates were confirmed. We observed a higher incidence of SLB symptoms on the oldest leaves compared with younger leaves. The maximum and minimum temperatures for in vitro mycelial growth and conidial germination (%) of S. vesicarium were 20 to 30°C and 5°C, respectively. Sixteen fungicides were tested for their effectiveness to reduce the mycelial growth and conidial germination of S. vesicarium in vitro. Boscalid plus pyraclostrobin, fluopyram, fluxapyroxad, and fluxapyroxad plus pyraclostrobin were highly effective at reducing mycelial growth and conidial germination in S. vesicarium. However, strobilurin fungicides (azoxystrobin and kresoxim-methyl) commonly used in Welsh onion production in Sanxing were ineffective. This study discusses the emergence of SLB caused by S. vesicarium in the foliar disease complex affecting Welsh onion and the management of the disease using fungicides with different modes of action in Taiwan. The research will support the sustainable management of SLB in Sanxing, Taiwan; however, further field assessments of the fungicides are warranted.

Marker-Assisted Development and Evaluation of Monogenic Lines of Rice cv. Kaohsiung 145 Carrying Blast Resistance Genes.

Rice blast is a serious threat to global rice production. Large-scale and long-term cultivation of rice varieties with a single blast resistance gene usually leads to breakdown of resistance. To effectively control rice blast in Taiwan, marker-assisted backcrossing was conducted to develop monogenic lines carrying different blast resistance genes in the genetic background of an elite japonica rice cultivar, Kaohsiung 145 (KH145). Eleven International Rice Research Institute (IRRI)-bred blast-resistant lines (IRBLs) showing broad-spectrum resistance to local Pyricularia oryzae isolates were used as resistance donors. Sequencing analysis revealed that the recurrent parent, KH145, does not carry known resistance alleles at the target Pi2/9, Pik, Pita, and Ptr loci. For each IRBL × KH145 cross, we screened 21 to 370 (average of 108) plants per generation from the BC1F1 to BC3F1/BC4F1 generation. A total of 1,499 BC3F2/BC4F2 lines carrying homozygous resistance alleles were selected and self-crossed for four to six successive generations. The derived lines were also evaluated for background genotype using genotyping by sequencing, for blast resistance under artificial inoculation and natural infection conditions, and for agronomic performance in multiple field trials. In Chiayi and Taitung blast nurseries in 2018 to 2020, Pi2, Pi9, and Ptr conferred high resistance, Pi20 and Pik-h moderate resistance, and Pi1, Pi7, Pik-p, and Pik susceptibility to leaf blast; only Pi2, Pi9, and Ptr conferred effective resistance against panicle blast. The monogenic lines showed agronomic traits, yield, and grain quality similar to those of KH145, suggesting the potential of growing a mixture of lines to achieve durable resistance in the field.

Soil Is Not a Reservoir for Phellinus noxius.

Phellinus noxius causes brown root rot (BRR) of diverse trees. Basidiospores and diseased host tissues have been recognized as important sources of P. noxius inoculum. This study aimed to understand whether P. noxius could occur or survive in soil without host tissues in the natural environment. Soil was sampled before and after the removal of diseased trees at eight BRR infection sites (total of 44 samples). No P. noxius colonies were recovered in soil plating assays, suggesting that no or little viable P. noxius resided in the soil. To know whether P. noxius could disseminate from decayed roots to the surrounding soil, rhizosphere and non-rhizosphere soils were sampled from another two infection sites. Although P. noxius DNA was detectable with specific primers, no P. noxius could be isolated, even from the rhizosphere soils around decayed roots covered with P. noxius mycelial mats. The association between viable P. noxius and the presence of its DNA was also investigated using field soil mixed with P. noxius arthrospores. After P. noxius was exterminated by flooding or fumigation treatment, its DNA remained detectable for a few weeks. The potential of onsite soil as an inoculum was tested using the highly susceptible loquat (Eriobotrya japonica). Loquats replanted in an infection site that had been cleaned up by simply removing the diseased stump and visible residual roots remained healthy for a year. Taken together, P. noxius is not a soilborne pathogen, and diseased host tissues should be the focus of field sanitation and detection for BRR.

Invasion and Colonization Pattern of Fusarium fujikuroi in Rice.

Bakanae disease in rice can cause abnormal elongation of the stem and leaves, development of adventitious roots, a larger leaf angle, and even death. Little is known about the infection, colonization, and distribution of Fusarium fujikuroi in rice plants across different growth stages. In this study, microscopic observation and quantitative real-time PCR were combined to investigate the pathogenesis of bakanae, using artificially inoculated seedlings of a susceptible rice cultivar, Zerawchanica karatals (ZK), a resistant cultivar, Tainung 67 (TNG67), naturally infected adult field plants (cultivars Kaohsiung 139, Taikeng 2, and Tainan 11), and an F. fujikuroi isolate expressing green fluorescent protein. In rice seedlings, F. fujikuroi hyphae were found to directly penetrate the epidermis of basal stems and roots, then extend inter- and intracellularly to invade the vascular bundles. Occlusion of vascular bundles and radial hyphal expansion from vascular bundles to surrounding parenchyma were observed in adult plants. Analysis of consecutive 3-cm segments of the whole plant revealed that F. fujikuroi was largely confined to the embryo, basal stem, and basal roots in seedlings, and distributed unevenly in the lower aerial parts (including nodes and internodes) of adult plants. The elongation and development of adventitious roots did not necessarily correlate with the amount of F. fujikuroi in diseased plants. Treatment of rice seeds with gibberellic acid-3 (GA3) at 0.5 mg/liter resulted in significantly more elongation of ZK than TNG67 seedlings, suggesting that the susceptibility of ZK to bakanae is associated with its higher sensitivity to GA3.

First Report of Neopestalotiopsis rosae Causing Leaf Blight and Crown Rot on Strawberry in Taiwan.

For the past 30 years, the most predominant strawberry cultivar in Taiwan has been 'Taoyuan No. 1', which produces fruit with rich flavor and aroma but is highly susceptible to anthracnose (Chung et al. 2019). Because epidemics of anthracnose became more destructive, farmers switched to an anthracnose-tolerant cultivar 'Xiang-Shui' (~50% and ~80% of the cultivation area in 2018 and 2019, respectively). Since 2018, severe leaf blight and crown rot symptoms have been observed all year in 'Xiang-Shui' in Miaoli, Nantou, Hsinchu, Taipei, Taoyuan, and Chiayi Counties. The disease became more prevalent and severe during 2019 to 2020 and caused up to 30% plant loss after transplanting. Symptoms appeared as brown necrotic lesions with black acervuli on leaves, slightly sunken dark-brown necrosis on stolons, and sunken reddish-brown necrosis on fruit. The diseased crown tissue showed marbled reddish-brown necrosis with a dark-brown margin, and plants with severe crown rot usually showed reddish-brown discoloration on leaves (the leaves initially turned reddish-brown between the veins and could become entirely scorched at later stages). To isolate the causal agent, small fragments of diseased leaves, crowns, stolons, and fruits were surface-disinfested with 0.5% sodium hypochlorite for 30 seconds, rinsed with sterile water then placed on 1.5% water agar. Single hyphal tips extended from tissues were transferred to potato dextrose agar and cultured for 7 days at 25°C under a 12-h/12-h photoperiod. Total 20 isolates were obtained from diseased leaves, crowns, stolons, and fruits. Colonies were white with cottony aerial mycelium, irregular margins, and black acervuli distributed in concentric rings. Conidia were fusiform to ellipsoid (five cells) with one basal appendage and three or four (usually three) apical appendages. From colony and conidial morphology, the causal agent was identified as Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) region, ß-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) of three isolates (ML1664 from diseased crown tissue collected in Hsinchu County; ML2147 and ML2411 from diseased leaves collected in Miaoli County) were sequenced (GenBank nos. MT469940 to MT469948). All three isolates clustered with the ex-type strain of Neopestalotiopsis rosae in the multilocus (ITS+TUB+TEF-1α) phylogenetic tree. To fulfill Koch's postulates, spore suspensions of ML1664 and ML2147 at 1×106 conidia/mL were used to spray-inoculate 'Xiang-Shui' seedlings at the 3 to 4 leaf stage until run-off (two trials, five seedlings per trial). Inoculated plants were put in a plastic bag (> 90% RH) at 25°C under a 12-h/12-h photoperiod. After 10-14 days, 80% of inoculated plants showed leaf or crown symptoms similar to those in the field. Control plants sprayed with sterile water showed no symptoms (4-5 seedlings per trial). The fungi were re-isolated from necrotic lesions with 100% frequency (n ≥ 3 isolates per trial), and morphological characters and ITS sequences were identical to the original ones. This is the first report of N. rosae causing leaf blight and crown rot in strawberry in Taiwan. N. rosae and N. clavispora have been reported as new threats to strawberry in several other countries (Rebollar-Alviter 2020; Gilardi 2019). Clarification of the pathogen provides a basis for developing strategies to control the emerging disease. Further studies are needed to evaluate the resistance/susceptibility of major strawberry cultivars and the fungicide sensitivity of the pathogen.

First Report of Xanthomonas fragariae Causing Angular Leaf Spot on Strawberry (Fragaria x ananassa) in Taiwan.

Angular leaf spot of strawberry, considered an A2 quarantine pest by the European and Mediterranean Plant Protection Organization (EPPO 2019), is an important bacterial disease in many regions. Since 2017, symptoms similar to angular leaf spot were observed in several strawberry cultivars including 'Taoyuan No. 1' and 'Xiang-Shui'. Early symptoms were angular, water-soaked lesions on the abaxial leaf surface, and later, reddish-brown irregular spots and coalesced lesions developed on the adaxial surface. In the humid conditions, sticky bacterial ooze exuding from lesions was observed. To isolate the causal agent, leaves showing water-soaked lesions were surface sterilized, cut into small pieces and soaked in 5 ml sterile water for at least 15 min. The supernatant from the cut-up pieces was serially diluted followed by spreading on sucrose peptone agar (SPA) (Hayward 1960). After incubating at 20°C for 4-5 days, single colonies grown on SPA were transferred to a new SPA plate and cultured at 20°C until colonies appeared. The yellow, glossy and mucoid colonies, which resembled the colony morphology of Xanthomonas fragariae, were selected as candidates for further confirmation. First, bacterial DNA of four candidate isolates, B001, B003 and B005 from Miaoli County and B004 from Taoyuan City, was PCR amplified with X. fragariae-specific primers: XF9/XF12 (Roberts et al. 1996) and 245A/B and 295A/B (Pooler et al. 1996). All four isolates could be detected by XF9/XF12 primer. Furthermore, isolates B003 and B004 could be detected by both 245A/B and 295A/B primers, while B001 and B005 could be detected by 295A/B only. Next, DNA gyrase subunit B (gyrB) was PCR amplified with the primers XgyrB1F/XgyrB1R (Young et al. 2008). The gyrB sequences of these four isolates were deposited in GenBank with accession numbers MT754942 to MT754945. The gyrB phylogenetic tree was constructed based on Bayesian inference analysis and maximum likelihood analysis. The gyrB sequences of the four isolates from Taiwan clustered in the clade containing the type strain of X. fragariae ICMP5715, indicating that they belong to X. fragariae. B001 and B005 formed a sub-group separated from B003 and B004, suggesting genetic differences between these isolates. To fulfill Koch's postulates, the abaxial surface of strawberry leaves were syringe infiltrated (KJP Silva et al., 2017) or wounded inoculated (Wang et al., 2017) with bacterial suspensions (final OD600 = 1.0-2.0) prepared from colonies of B001 and B003 washed from SPA plates. Inoculated plants were enclosed in a plastic bag (> 90% RH) at 25/20°C (day/night) under a 12-h/12-h photoperiod. After 7-14 days, water-soaked lesions similar to those observed in the field were developed on all inoculated leaves. The bacteria were successfully re-isolated from lesions of inoculated leaves and confirmed by specific primers XF9/XF12, 245A/B and 295A/B. We also found that the disease commonly occurs in the strawberry fields/nurseries with sprinkler irrigation during winter or early spring, and was particularly serious in the windward side or near riverside. To our knowledge, this is the first report of X. fragariae causing angular leaf spot on strawberry in Taiwan. Currently, the disease only occurs severely in certain regions, but establishment of effective management strategies will be needed to prevent spreading of this disease and potential economic loss in the future.

In Vitro and in Planta Evaluation of Trichoderma asperellum TA as a Biocontrol Agent Against Phellinus noxius, the Cause of Brown Root Rot Disease of Trees.

Brown root rot (BRR), caused by the white rot fungus Phellinus noxius, is an epidemic disease of diverse broadleaved and coniferous tree species in many tropical and subtropical regions. Flooding and trenching control measures are difficult to implement, and chemical controls can have an adverse impact on ecosystems. Previous studies have provided in vitro evidence for the potential use of Trichoderma spp. for biocontrol of BRR. Here, we analyzed the in vitro antagonistic and mycoparasitic abilities of four Trichoderma spp. isolates against four P. noxius isolates in dual culture and Ficus microcarpa wood blocks. A convenient inoculation system based on root inoculation of a highly susceptible loquat (Eriobotrya japonica) with P. noxius-colonized wheat-oat grains was developed to examine the effect of Trichoderma treatment in planta. Preventive application of Trichoderma asperellum TA, the isolate showing high antagonistic activity in vitro, was effective in preventing and delaying the wilting of P. noxius-inoculated loquat cuttings in greenhouse trials. To understand the specific niche in which T. asperellum TA interacts with P. noxius, KOH-aniline blue fluorescence microscopy was used to investigate the colonization of loquat roots by P. noxius and/or T. asperellum TA. Dilution plating assays were also conducted to quantify Trichoderma populations in the rhizosphere and potting mix. T. asperellum TA was able to robustly establish in the rhizosphere and potting mix but with scarce root penetration limited to the superficial layer. We discuss the timing and strategy for applying antagonistic Trichodema sp. on living trees or in BRR-infested areas for BRR management.

Determinants of Virulence and In Vitro Development Colocalize on a Genetic Map of Setosphaeria turcica.

Generating effective and stable strategies for resistance breeding requires an understanding of the genetics of host-pathogen interactions and the implications for pathogen dynamics and evolution. Setosphaeria turcica causes northern leaf blight (NLB), an important disease of maize for which major resistance genes have been deployed. Little is known about the evolutionary dynamics of avirulence (AVR) genes in S. turcica. To test the hypothesis that there is a genetic association between avirulence and in vitro development traits, we (i) created a genetic map of S. turcica, (ii) located candidate AVRHt1 and AVRHt2 regions, and (iii) identified genetic regions associated with several in vitro development traits. A cross was generated between a race 1 and a race 23N strain, and 221 progeny were isolated. Genotyping by sequencing was used to score 2,078 single-nucleotide polymorphism markers. A genetic map spanning 1,981 centimorgans was constructed, consisting of 21 linkage groups. Genetic mapping extended prior evidence for the location and identity of the AVRHt1 gene and identified a region of interest for AVRHt2. The genetic location of AVRHt2 colocalized with loci influencing radial growth and mycelial abundance. Our data suggest a trade-off between virulence on Ht1 and Ht2 and the pathogen's vegetative growth rate. In addition, in-depth analysis of the genotypic data suggests the presence of significant duplication in the genome of S. turcica.

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees.

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.

Genetic mapping of the qSBN7 locus, a QTL controlling secondary branch number per panicle in rice.

Secondary branch number (SBN) is an important component affecting spikelet number per panicle (SPP) and yield in rice. During recurrent backcross breeding, four BC2F4 populations derived from the high-yield donor parent IR65598-112-2 and the recurrent parent Tainan 13 (a local japonica cultivar) showed discontinuous variations of SPP and SBN within populations. Genetic analysis of 92 BC2F4 individuals suggested that both SPP and SBN are controlled by a single recessive allele. Two parents and 37 BC2F4 individuals showing high- and low-SBN type phenotypes were analyzed by restriction-site associated DNA sequencing (RAD-seq). Based on 2,522 reliable SNPs, the qSBN7 was mapped to a distal region of the long arm of chromosome 7. Trait-marker association analysis with an additional 166 high-SBN type BC2F4 individuals and 8 newly developed cleaved amplified polymorphic sequence markers further delimited the qSBN7 locus to a 601.4-kb region between the markers SNP2788 and SNP2849. Phenotype evaluation of two BC2F5 backcross inbred lines revealed that qSBN7 increased SPP by 83.2% and SBN by 61.0%. The qSBN7 of IR65598-112-2 could be used for improving reproductive sink capacity in rice.

A remorin gene is implicated in quantitative disease resistance in maize.

KEY MESSAGE: Quantitative disease resistance is used by plant breeders to improve host resistance. We demonstrate a role for a maize remorin ( ZmREM6.3 ) in quantitative resistance against northern leaf blight using high-resolution fine mapping, expression analysis, and mutants. This is the first evidence of a role for remorins in plant-fungal interactions. Quantitative disease resistance (QDR) is important for the development of crop cultivars and is particularly useful when loci also confer multiple disease resistance. Despite its widespread use, the underlying mechanisms of QDR remain largely unknown. In this study, we fine-mapped a known quantitative trait locus (QTL) conditioning disease resistance on chromosome 1 of maize. This locus confers resistance to three foliar diseases: northern leaf blight (NLB), caused by the fungus Setosphaeria turcica; Stewart's wilt, caused by the bacterium Pantoea stewartii; and common rust, caused by the fungus Puccinia sorghi. The Stewart's wilt QTL was confined to a 5.26-Mb interval, while the rust QTL was reduced to an overlapping 2.56-Mb region. We show tight linkage between the NLB QTL locus and the loci conferring resistance to Stewart's wilt and common rust. Pleiotropy cannot be excluded for the Stewart's wilt and the common rust QTL, as they were fine-mapped to overlapping regions. Four positional candidate genes within the 243-kb NLB interval were examined with expression and mutant analysis: a gene with homology to an F-box gene, a remorin gene (ZmREM6.3), a chaperonin gene, and an uncharacterized gene. The F-box gene and ZmREM6.3 were more highly expressed in the resistant line. Transposon tagging mutants were tested for the chaperonin and ZmREM6.3, and the remorin mutant was found to be more susceptible to NLB. The putative F-box is a strong candidate, but mutants were not available to test this gene. Multiple lines of evidence strongly suggest a role for ZmREM6.3 in quantitative disease resistance.

The Genetic Structure, Virulence, and Fungicide Sensitivity of Fusarium fujikuroi in Taiwan.

The rice disease bakanae, caused by Fusarium fujikuroi Nirenberg, has been present in Taiwan for over a century. To better understand the genetic diversity and structure of F. fujikuroi, a set of 16 polymorphic simple sequence repeat (SSR) markers were newly developed and used to analyze 637 F. fujikuroi isolates collected in 14 cities or counties around Taiwan from 1996 to 2013. On the basis of Bayesian clustering, the isolates were classified into four highly differentiated clusters: cluster B likely derived from the more widespread and genetically diversified clusters A or C, and cluster D was restricted to four cities or counties and may have been introduced from unknown sources genetically distinct from clusters A, B, and C. The coexistence of both mating types (MAT1-1:MAT1-2 = 1:1.88) and the highly diversified vegetative compatibility groups (VCG) (16 VCG among the 21 assessed isolates) suggest the likelihood of sexual reproduction in the field. However, the biased mating type ratios and linkage disequilibrium in the population suggest nonrandom mating between individuals. A significant pattern of isolation by distance was also detected, which implies a geographical restricted gene flow and low dissemination ability of F. fujikuroi. Evaluation of 24 representative isolates on eight rice varieties revealed differential levels of virulence, however no clear pattern of specific variety x isolate interaction was observed. Investigations of the differences in virulence and fungicide sensitivity between 8 early isolates (1998 and 2002) and 52 recent isolates (2012) indicate the evolution of increased resistance to the fungicide prochloraz in F. fujikuroi in Taiwan.