Inter- and Intra-Specific Variations in Phenotypic Traits of Pectobacterium Strains Isolated from Diverse Eudicots and Monocots in Taiwan.

Pectobacterium spp. are phytopathogenic bacteria whose phylogeny has been continuously revised throughout the years. Previous studies on Pectobacterium's phenotypic diversity often analyzed strains obtained from specific crops or adopted outdated Pectobacterium classification systems. Therefore, a current perspective on trait variations in Pectobacterium species or strains infecting more diverse plant species is limited. This study conducted phylogenetic and phenotypic analyses on strains isolated from eight eudicot and four monocot families in Taiwan. Phylogenetic analysis on 78 strains identified six recognized species, namely, P. brasiliense, P. aroidearum, P. actinidiae, P. colocasium, P. carotovorum, and P. versatile. Among these, the first two were the most predominant species. Patterns suggesting varying host preferences among bacterial species were detected; most P. aroidearum strains were isolated from monocots, whereas P. brasiliense and P. actinidiae tended to exhibit preferences for eudicots. Physiological tests and Biolog analyses conducted on representative strains of each species revealed great within-species phenotypic variations. Despite these strain-level variations, a combination of indole production and phosphatase activity tests was capable of distinguishing all representative strains of P. brasiliense from those of other identified species. Inoculation assays on potato, bok choy, calla lily, and onion showed inter- and intra-specific heterogeneities in the tested strains' maceration potentials. Virulence patterns across Pectobacterium species and strains differed depending on the inoculated host. Altogether, the findings from this work expand the understanding of Pectobacterium's phenotypic diversity and provide implications for pathogen identification and management.

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.

First Report of Binucleate Rhizoctonia AG-L Causing Root and Stem Rot of Wishbone Flower (Torenia fournieri) in Taiwan.

Wishbone flower (Torenia fournieri L.) is a common ornamental plant for flower bed in Taiwan. In August 2018, root and stem rot of wishbone flower occurred on the flower bed in the campus of National Chung Hsing University, Taichung city, with 100% incidence. Symptoms were dark brown discoloration of basal stems and brown necrosis of roots. Lesions from base of stems were excised into 5 mm long fragments, which were then surface sterilized in 1% sodium hydrochloride for 1 min, rinsed in sterile distilled water, dried on filter paper and thereafter placed onto 2% water agar. After 24 h, hyphae characteristic of Rhizoctonia (Sneh et al. 1991) appeared and dominated in every isolation. Hyphal tips were transferred to potato dextrose agar (PDA). After 5 days of incubation at 28°C, characteristic brown colonies of Rhizoctonia (Sneh et al. 1991) were observed. Hyphal width was 4.29±0.52 µm. No sclerotia were visibly present after 21 days of growth on PDA at 28°C. Hyphae were stained by 0.3% safranin-O and 1% KOH. There were two nuclei in each hyphal compartment, suggesting a binucleate Rhizoctonia fungus. ITS sequence has been used as the best tool to identify specific anastomosis group (AG) of Rhizoctonia as shown by Sharon et al. (2006, 2008). ITS sequence was amplified using the primers Bd1a and ITS4 (Goka et al. 2009; White et al. 1990). Blast search analysis of this acquired sequence (acc. no. LC498494) revealed the highest similarity (98.75 to 99.83%) with the reference sequences (acc. nos. AB286934, AB286933, and AB196653) of binucleate Rhizoctonia AG-L, namely Ceratobasidium sp. AG-L. Pathogenicity test was carried out using seedlings of 4-week-old wishbone flower each grown in a pot of 6.35 cm diameter. To prepare the inoculum, a PDA agar block (6 mm in diameter) excised from the growing front of 5-day-old colony was transferred into a flask with 200 ml of potato dextrose broth (PDB) incubated in a shaker at 26°C and 120 rpm for 6 days. The PDB broth was then blended into slurry. Ten pots each with a seedling were inoculated by pouring 50 ml of slurry onto the potting medium. Five pots were served as the controls by pouring PDB only. Pots were maintained in a greenhouse (26 to 33°C). Three days after inoculation, all inoculated plants exhibited symptom of root and stem rot. The same fungus was reisolated and confirmed by sequencing rDNA-ITS. This is the first report of root and stem rot of wishbone flower caused by binucleate Rhizoctonia AG-L in Taiwan and in the world. Although this is the second cases, since Wang and Hsieh (1993), for binucleate Rhizoctonia AG-L to be pathogenic, this study shows that this fungus has the potential to cause damages and is worth of further investigations.

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.