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.

Identification of qBK2.1, a novel QTL controlling rice resistance against Fusarium fujikuroi.

BACKGROUND: Bakanae disease caused by Fusarium fujikuroi is an increasing threat to rice production. The infected plants show symptoms such as elongation, slenderness, chlorosis, a large leaf angle, and even death. Bakanae disease is traditionally managed by seed treatment. However, fungicide-resistant F. fujikuroi isolates have emerged in several Asian areas, including Taiwan. This study aimed to identify new bakanae resistance quantitative trait loci (QTLs) and provide molecular markers to assist future breeding. RESULTS: A population of F2:9 recombinant inbred lines (RILs) was derived from the cross between an elite japonica Taiwanese cultivar 'Taikeng 16 (TK16)' and an indica variety 'Budda'. 'Budda' was found highly resistant to all 24 representative isolates of the F. fujikuroi population in Taiwan. For the RIL population, 6,492 polymorphic single nucleotide polymorphisms (SNPs) spanning the rice genome were obtained by genotyping-by-sequencing (GBS) technique, and the disease severity index (DSI) was evaluated by inoculation with a highly virulent F. fujikuroi isolate Ff266. Trait-marker association analysis of 166 RILs identified two QTLs in 'Budda'. qBK2.1 (21.97-30.15 Mb) is a novel and first bakanae resistance QTL identified on chromosome 2. qBK1.8 (5.24-8.66 Mb) partially overlaps with the previously reported qBK1.3 (4.65-8.41 Mb) on chromosome 1. The log of odds (LOD) scores of qBK1.8 and qBK2.1 were 4.75 and 6.13, accounting for 4.9% and 8.1% of the total phenotypic variation, respectively. 64 RILs carrying both qBK1.8 and qBK2.1 showed lower DSI (7%) than the lines carrying only qBK1.8 (15%), only qBK2.1 (13%), or none of the two QTLs (21%). For the future application of identified QTLs, 11 KBioscience competitive allele-specific PCR (KASP) markers and 3 insertion-deletion (InDel) markers were developed. CONCLUSIONS: Compared to other important rice diseases, knowledge of bakanae resistance has been insufficient, which limited the development and deployment of resistant cultivars. The discovery of qBK2.1 has provided a new source of bakanae resistance. The resistant RILs inheriting good plant type, good taste, and high yield characteristics from 'TK16' can be used as good resistance donors. Our newly developed markers targeting qBK2.1 and qBK1.8 can also serve as an important basis for future fine-mapping and resistance breeding.

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.

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.

Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants.

Anthracnose is a major disease of strawberry that seriously impacts the strawberry industry. To prevent the spread of anthracnose through symptomless plants, it is important to detect pathogenic Colletotrichum spp. at the latent infection stage in the nursery. Previous PCR-based methods developed for the diagnosis or detection of Colletotrichum acutatum and Colletotrichum gloeosporioides have used primers targeting the internal transcribed spacer region of ribosomal DNA, ß-tubulin gene, or mating type gene. In this study, to specifically detect Colletotrichum siamense and Colletotrichum fructicola, the most predominant and virulent Colletotrichum species causing strawberry anthracnose in Taiwan, we conducted a comparative genomics analysis of 29 Colletotrichum spp. and identified a non-conserved 1157-bp intergenic region suitable for designing specific primers for a nested PCR assay. In silico analysis and actual tests suggested that the new nested PCR assay could detect pathogenic C. siamense and C. fructicola, but not other strawberry pathogens (Botrytis sp., Fusarium spp., Neopestalotiopsis rosae, and Phytophthora sp.) or ubiquitous saprophytes (Fusarium spp. and Trichoderma spp.). The inner to outer primer ratio was optimized to 1:10 to eliminate unexpected bands and enhance the signal. The assay could detect as little as 1 pg of C. siamense genomic DNA, which corresponds to ~15 cells. Application of the new detection assay on 747 leaf samples collected from 18 strawberry nurseries in 2019 and 2020 showed that an average of 20% of strawberry mother plants in Taiwan were latently infected by C. siamense or C. fructicola. The newly developed assay is being applied to facilitate the production of healthy strawberry runner plants in Taiwan.

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.

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.

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.

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.

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.

Transcriptome Analysis of Early Defenses in Rice against Fusarium fujikuroi.

BACKGROUND: Bakanae is a seedborne disease caused by Fusarium fujikuroi. Rice seedlings emerging from infected seeds can show diverse symptoms such as elongated and slender stem and leaves, pale coloring, a large leaf angle, stunted growth and even death. Little is known about rice defense mechanisms at early stages of disease development. RESULTS: This study focused on investigating early defenses against F. fujikuroi in a susceptible cultivar, Zerawchanica karatals (ZK), and a resistant cultivar, Tainung 67 (TNG67). Quantitative PCR revealed that F. fujikuroi colonizes the root and stem but not leaf tissues. Illumina sequencing was conducted to analyze the stem transcriptomes of F. fujikuroi-inoculated and mock-inoculated ZK and TNG67 plants collected at 7 days post inoculation (dpi). More differentially expressed genes (DEGs) were identified in ZK (n = 169) than TNG67 (n = 118), and gene ontology terms related to transcription factor activity and phosphorylation were specifically enriched in ZK DEGs. Among the complex phytohormone biosynthesis and signaling pathways, only DEGs involved in the jasmonic acid (JA) signaling pathway were identified. Fourteen DEGs encoding pattern-recognition receptors, transcription factors, and JA signaling pathway components were validated by performing quantitative reverse transcription PCR analysis of individual plants. Significant repression of jasmonate ZIM-domain (JAZ) genes (OsJAZ9, OsJAZ10, and OsJAZ13) at 3 dpi and 7 dpi in both cultivars, indicated the activation of JA signaling during early interactions between rice and F. fujikuroi. Differential expression was not detected for salicylic acid marker genes encoding phenylalanine ammonia-lyase 1 and non-expressor of pathogenesis-related genes 1. Moreover, while MeJA did not affect the viability of F. fujikuroi, MeJA treatment of rice seeds (prior to or after inoculation) alleviated and delayed bakanae disease development in susceptible ZK. CONCLUSIONS: Different from previous transcriptome studies, which analyzed the leaves of infected plants, this study provides insights into defense-related gene expression patterns in F. fujikuroi-colonized rice stem tissues. Twelve out of the 14 selected DEGs were for the first time shown to be associated with disease resistance, and JA-mediated resistance was identified as a crucial component of rice defense against F. fujikuroi. Detailed mechanisms underlying the JA-mediated bakanae resistance and the novel defense-related DEGs are worthy of further investigation.

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.

Diversity and pathogenicity of Colletotrichum species causing strawberry anthracnose in Taiwan and description of a new species, Colletotrichum miaoliense sp. nov.

Strawberry is a small fruit crop with high economic value. Anthracnose caused by Colletotrichum spp. poses a serious threat to strawberry production, particularly in warm and humid climates, but knowledge of pathogen populations in tropical and subtropical regions is limited. To investigate the diversity of infectious agents causing strawberry anthracnose in Taiwan, a disease survey was conducted from 2010 to 2018, and Colletotrichum spp. were identified through morphological characterization and multilocus phylogenetic analysis with internal transcribed spacer, glyceraldehyde 3-phosphate dehydrogenase, chitin synthase, actin, beta-tubulin, calmodulin, and the intergenic region between Apn2 and MAT1-2-1 (ApMAT). Among 52 isolates collected from 24 farms/nurseries in Taiwan, a new species, Colletotrichum miaoliense sp. nov. (6% of all isolates), a species not previously known to be associated with strawberry, Colletotrichum karstii (6%), and three known species, Colletotrichum siamense (75%), Colletotrichum fructicola (11%), and Colletotrichum boninense (2%), were identified. The predominant species C. siamense and C. fructicola exhibited higher mycelial growth rates on potato dextrose agar and caused larger lesions on wounded and non-wounded detached strawberry leaves. Colletotrichum boninense, C. karstii, and C. miaoliense only caused lesions on wounded leaves. Understanding the composition and biology of the pathogen population will help in disease management and resistance breeding.

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.

A Novel Variation in the FRIZZLE PANICLE (FZP) Gene Promoter Improves Grain Number and Yield in Rice.

Secondary branch number per panicle plays a crucial role in regulating grain number and yield in rice. Here, we report the positional cloning and functional characterization for SECONDARY BRANCH NUMBER7 (qSBN7), a quantitative trait locus affecting secondary branch per panicle and grain number. Our research revealed that the causative variants of qSBN7 are located in the distal promoter region of FRIZZLE PANICLE (FZP), a gene previously associated with the repression of axillary meristem development in rice spikelets. qSBN7 is a novel allele of FZP that causes an ∼56% decrease in its transcriptional level, leading to increased secondary branch and grain number, and reduced grain length. Field evaluations showed that qSBN7 increased grain yield by 10.9% in a temperate japonica variety, TN13, likely due to its positive effect on sink capacity. Our findings suggest that incorporation of qSBN7 can increase yield potential and improve the breeding of elite rice varieties.

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.

Genome-wide association mapping of gene loci affecting disease resistance in the rice-Fusarium fujikuroi pathosystem.

BACKGROUND: Rice bakanae disease has emerged as a new threat to rice production. In recent years, an increase in the occurrence and severity of bakanae disease has been reported in several areas in Asia. Although bakanae disease affects rice yield and quality, little is known about the genetics of bakanae resistance in rice. The lack of large-scale screens for bakanae resistance in rice germplasm has also limited the development and deployment of resistant varieties. RESULTS: A genome-wide association study (GWAS) was conducted to identify genes/loci conferring bakanae resistance in rice. A total of 231 diverse accessions from Rice Diversity Panel 1 (RDP1) were inoculated with a highly virulent Taiwanese Fusarium fujikuroi isolate and assessed for resistance using two parameters: (1) disease severity index based on visual rating and (2) colonization rate determined by reisolation of F. fujikuroi from the basal stems of infected rice seedlings. We identified 14 quantitative trait loci (QTLs) (10 for disease severity and 4 for colonization rate), including 1 mapped for both parameters. A total of 206 candidate genes were identified within the 14 QTLs, including genes encoding leucine-rich repeat (LRR)-containing and NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) proteins, hormone-related genes, transcription factor genes, ubiquitination-related genes, and oxidase/oxidoreductase genes. In addition, a candidate QTL (qBK1.7) that co-localized with the previously identified QTLs qBK1 and qFfR1, was verified by linkage analysis using a population of 132 recombinant inbred lines derived from IR64 x Nipponbare. GWAS delineated qBK1.7 to a region of 8239 bp containing three genes. Full-length sequencing across qBK1.7 in 20 rice accessions revealed that the coding regions of two LRR-containing genes Os01g0601625 and Os01g0601675 may be associated with bakanae resistance. CONCLUSIONS: This study facilitates the exploitation of bakanae resistance resources in RDP1. The information on the resistance performance of 231 rice accessions and 14 candidate QTLs will aid efforts to breed resistance to bakanae and uncover resistance mechanisms. Quantification of the level of F. fujikuroi colonization in addition to the conventional rating of visual symptoms offers new insights into the genetics of bakanae resistance.