First Report of Fusarium oxysporum Causing Coriander Wilt Disease in North China

Coriander or cilantro (Coriandrum sativum L.) is extensively used as a fresh herb in China. During the summer of 2019 in Tianjin, China, coriander plants showed a previously unknown wilt in commercial fields. In severely infected fields, approximately 85% of the plants at vegetative stages were stunted, chlorotic, wilted or dead. Vascular tissues of the main stem and root were discolored. Soil samples were collected from five fields with a history of severe wilt disease in Tianjin since the fall of 2019. Seeds of coriander cultivar “ansemi” were sown in soil from the five fields, and cultured in a greenhouse at 22°C/20°C (12 h /12 h, light/dark) and 75% relative humidity. After 50 days’ cultivation, infected seedlings exibited similar symptoms to those of plants in the field. Twenty symptomic seedlings from each of the collected field soil samples were harvested and washed for 3 min under running water. Then the vascular tissue fragments (3 mm2) of stem and root at the boundary of the symptomatic area were excised, and placed on improved Komada’s medium, which is selective for Fusarium sp. (Komada 1975). After incubation at 25°C for 5 days in the dark, 70% of the isolates generated white to pale pink aerial hyphae on PDA (Fig. S1). Microconidia were single-celled, hyaline, non-septate and ovoid, and measured 5.6 to 14.1 μm long and 2.1 to 3.8 μm wide (n = 40). Macroconidia were three to five-septate, slightly curved at apex, and ranged from 11.2 to 38.6 μm long × 3.2 to 4.5 μm wide (n = 40). Based on morphological characteristics, these fungi were preliminarily identified as F. oxysporum (Leslie and Summerell 2006). For molecular identification, the ITS gene, TEF1-α gene and mtSSU gene of cultures from two representative single spored isolates XC02 and XC03 were amplified and sequenced (White et al. 1990; Carbone and Kohn 1999; Li et al. 1994). The sequences were submitted to GenBank (MT579855 and OK326765 for ITS; MT597425 and OK256882 for TEF1-α; MT587799 and OK330480 for mtSSU). BLASTn analysises indicated that the nucleotide sequences of the three loci of the two isolates were 99.8% to 100% homologous to sequences of F. oxysporum in the NCBI database and Fusarium MLST database. A multilocus phylogenetic tree was drawn via UPGMA analysis of the combined ITS, TEF1-α and mtSSU partial sequence data from Fusarium MLST database (Fig. S2). Pathogenicity of each isolate was tested on ten 15-day-old healthy coriander seedlings in each treatment according to the method of Yang et al. (2020). The first wilt symptoms developed 3 days after inoculation, and 7 to 10 days after inoculation 90% to 100% of the plants were dead. Control plants remained healthy. To fulfill the Koch's postulates, F. oxysporum were reisolated from the diseased tissues and verified based on morphology and sequencing as described above. The experiments were repeated twice with similar results. F. oxysporum has been reported to cause coriander wilt disease in India, Argentina, California (Koike and Gordon 2005), and Italy (Gilardi et al. 2019). To our knowledge, this is the first report of F. oxysporum causing coriander wilt disease in north China. Compared with F. equiseti in the previous report (Yang et al. 2020), F. oxysporum isolates possessed higher separation frequence and stronger pathogenicity to coriander seedlings. Thus attentions should be taken on F. oxysporum causing coriander wilt diseases when developing effective management strategies in north China.

Multi-locus sequence typing for species/serovar identification of clinical isolates of Leptospira spp

Leptospirosis is an emerging zoonotic disease endemic in Kerala and close monitoring of emerging serovars is essential to adopt appropriate control strategies. Multi-Locus Sequence Typing (MLST) was reported to be less expensive compared to other cumbersome methods like whole genome sequencing. The present study was conducted to obtain isolates of Leptospira from infected animals and rats and for the identification of serovars using MLST. A total of 205 blood samples (dog, cat, cattle, goat), 43 urine samples (dog, cattle) and post-mortem kidney samples from various animals such as dog (n=12), cattle (n=2) and rat (n=25) were collected and subjected to polymerase chain reaction (PCR) using G1/G2 primers to identify the pathogenic Leptospira. Fifteen samples were found to be positive, these samples when inoculated in the Ellinghausen- McCullough-Johnson-Harris (EMJH) semi-solid medium to obtain ten isolates. These ten isolates were further subjected to secY, icdA and GyraseB PCR and sequenced. The obtained sequences were analysed using BLAST and were fed into specified MLST database of Leptospira scheme-3, the allelic profile and sequence type were generated. The MLST results obtained in the study indicated that the isolates S24 and S33 belonged to serovar Canicola, S40 and 47 were Sejroe and S19, S27, S55, S69 and S71 were Bataviae, Autumnalis, Pomona, Icterohaemorraghiae and Australis, respectively. It was concluded that MLST is a convenient method for identifying leptospiral serovars.

First Report of Fusarium Wilt Caused by Fusarium equiseti on Cabbage (Brassica oleracea var. capitate L.) in Korea

Cabbage (Brassica oleracea var. capitate L.) is an important vegetable crop that is widely cultivated throughout the world. In August 2019, wilting symptoms on cabbage (stunted growth, withered leaves, and wilted plants) were observed in a cabbage field of Pyeongchang, Gangwon Province, with an incidence of 5 to 10%. To identify the cause, symptomatic root tissue was excised, surface-sterilized with 70% ethanol, and rinsed thrice with sterile distilled water. The samples were dried on blotter paper, placed onto potato dextrose agar (PDA), and incubated at 25°C for 1 week. Five morphologically similar fungal isolates were sub-cultured and purified using the single spore isolation method (Choi et al. 1999). The fungus produced colonies with abundant, loosely floccose, whitish-brown aerial mycelia and pale-orange pigmentation on PDA. Macroconidia had four 4 to six 6 septa, a foot-shaped basal cell, an elongated apical cell, and a size of 20.2 to 31.8 × 2.2 to 4.1 μm (n = 30). No microconidia were observed. Chlamydospores were produced from hyphae and were most often intercalary, in pairs or solitary, globose, and frequently formed chains (6.2? to 11.7 μm, n = 10). Based on these morphological characteristics, the fungus was identified as Fusarium equiseti (Leslie and Summerell 2006). A representative isolate was deposited in the Korean Agricultural Culture Collection (KACC48935). For molecular characterization, portions of the translation elongation factor 1-alpha (TEF-1α) and second largest subunit of RNA polymerase II (RPB2) genes were amplified from the representative isolate using the primers pair of TEF-1α (O’Donnell et al. 2000) and GQ505815 (Fusarium MLST database), and sequenced. Searched BLASTn of the RPB2 sequence (MT576587) to the Fusarium MLST database showed 99.94% similarity to the F. incarnatum-equiseti species complex (GQ505850) and 98.85 % identity to both F. equiseti (GQ505599) and F. equiseti (GQ505772). Further, the TEF-1α sequence (MT084815) showed 100% identity to F. equiseti (KT224215) and 99.85% identity to F. equiseti (GQ505599), respectively. Therefore, the fungus was identified as F. equiseti based on morphological and molecular identification. For pathogenicity testing, a conidial suspension (1 × 106 conidia/ml) was prepared by harvesting macroconidia from 2-week-old cultures on PDA. Fifteen 4-week-old cabbage seedlings (cv. 12-Aadrika) were inoculated by dipping roots into the conidial suspension for 30 min. The inoculated plants were transplanted into a 50-hole plastic tray containing sterilized soil and maintained in a growth chamber at 25°C, with a relative humidity of >80%, and a 12-h/12-h light/dark cycle. After 4 days, the first wilt symptoms were observed on inoculated seedlings, and the infected plants eventually died within 1 to 2 weeks after inoculation. No symptoms were observed in plants inoculated with sterilized distilled water. The fungus was re-isolated from symptomatic tissues of inoculated plants and its colony and spore morphology were identical to those of the original isolate, thus confirming Koch's postulates. Fusarium wilt caused by F. equiseti has been reported in various crops, such as cauliflower in China, cumin in India, and Vitis vinifera in Spain (Farr and Rossman 2020). To our knowledge, this is the first report of F. equiseti causing Fusarium wilt on cabbage in Korea. It This disease poses a threat to cabbage production in Korea, and effective disease management strategies need to be developed.

New multilocus sequence typing primers to enable genotyping of AD hybrids within the Cryptococcus neoformans species complex

Although AD hybrids within the Cryptococcus neoformans species complex represent about 20% of the isolates identified in Europe, phylogenetic and population genetic studies are lacking due to the inability to use the standardized typing method. The aim of the present study was to design new molecular type specific primers in order to apply the standard ISHAM consensus multilocus sequence typing (MLST) scheme to AD hybrids. The new primers are able to specifically amplify VNI and VNIV alleles of the seven MLST loci in both haploid and diploid or aneuploid hybrid strains. This study forms the basis for future molecular epidemiology studies of AD hybrids. Lay abstract We designed and tested new specific primers to amplify the two alleles of each of the seven MLST loci in C. neoformans species complex hybrids. The sequences obtained from hybrids can be compared with those present in the Cryptococcus global MLST database for future molecular epidemiology studies.

Identification ofSalmonellafor public health surveillance using whole genome sequencing

In April 2015, Public Health England implemented whole genome sequencing (WGS) as a routine typing tool for public health surveillance ofSalmonella, adopting a multilocus sequence typing (MLST) approach as a replacement for traditional serotyping. The WGS derived sequence type (ST) was compared to the phenotypic serotype for 6,887 isolates ofS. entericasubspecies I, and of these, 6,616 (96%) were concordant. Of the 4% (n= 271) of isolates of subspecies I exhibiting a mismatch, 119 were due to a process error in the laboratory, 26 were likely caused by the serotype designation in the MLST database being incorrect and 126 occurred when two different serovars belonged to the same ST. The population structure ofS. entericasubspecies II–IV differs markedly from that of subspecies I and, based on current data, defining the serovar from the clonal complex may be less appropriate for the classification of this group. Novel sequence types that were not present in the MLST database were identified in 8.6% of the total number of samples tested (includingS. entericasubspecies I–IV andS. bongori) and these 654 isolates belonged to 326 novel STs. ForS. entericasubspecies I, WGS MLST derived serotyping is a high throughput, accurate, robust, reliable typing method, well suited to routine public health surveillance. The combined output of ST and serovar supports the maintenance of traditional serovar nomenclature while providing additional insight on the true phylogenetic relationship between isolates.

Identification and typing of Salmonella for public health surveillance using whole genome sequencing

In April 2015, Public Health England implemented whole genome sequencing (WGS) as a routine typing tool for public health surveillance of Salmonella, adopting a multilocus sequence typing (MLST) approach as a replacement for traditional serotyping. The WGS derived sequence type (ST) was compared to the phenotypic serotype for 6887 isolates of S. enterica subspecies I, and of these, 6616 (96%) were concordant. Of the 4% (n=271) of isolates of subspecies I exhibiting a mismatch, 119 were due to a process error in the laboratory, 26 were likely caused by the serotype designation in the MLST database being incorrect and 126 occurred when two different serovars belonged to the same ST. The population structure of S. enterica subspecies II-IV differs markedly from that of subspecies I and, based on current data, defining the serovar from the clonal complex may be less appropriate for the classification of this group. Novel sequence types that were not present in the MLST database were identified in 8.6% of the total number of samples tested (including S. enterica subspecies I-IV and S. bongori) and these 654 isolates belonged to 326 novel STs. For S. enterica subspecies I, WGS MLST derived serotyping is a high throughput, accurate, robust, reliable typing method, well suited to routine public health surveillance. The combined output of ST and serovar supports the maintenance of traditional serovar nomenclature while providing additional insight on the true phylogenetic relationship between isolates.

Identification and typing of Salmonella for public health surveillance using whole genome sequencing

In April 2015, Public Health England implemented whole genome sequencing (WGS) as a routine typing tool for public health surveillance of Salmonella, adopting a multilocus sequence typing (MLST) approach as a replacement for traditional serotyping. The WGS derived sequence type (ST) was compared to the phenotypic serotype for 6887 isolates of S. enterica subspecies I, and of these, 6616 (96%) were concordant. Of the 4% (n=271) of isolates of subspecies I exhibiting a mismatch, 119 were due to a process error in the laboratory, 26 were likely caused by the serotype designation in the MLST database being incorrect and 126 occurred when two different serovars belonged to the same ST. The population structure of S. enterica subspecies II-IV differs markedly from that of subspecies I and, based on current data, defining the serovar from the clonal complex may be less appropriate for the classification of this group. Novel sequence types that were not present in the MLST database were identified in 8.6% of the total number of samples tested (including S. enterica subspecies I-IV and S. bongori) and these 654 isolates belonged to 326 novel STs. For S. enterica subspecies I, WGS MLST derived serotyping is a high throughput, accurate, robust, reliable typing method, well suited to routine public health surveillance. The combined output of ST and serovar supports the maintenance of traditional serovar nomenclature while providing additional insight on the true phylogenetic relationship between isolates.

Exploring Diversity among NorwegianBorreliaStrains Originating fromIxodes ricinusTicks

Characterisation ofBorreliastrains fromIxodes ricinusticks is important in the epidemiological surveillance of vector-borne pathogens. Multilocus sequences analysis (MLSA) is a molecular genotyping tool with high discriminatory power that has been applied in evolutionary studies and for the characterisation ofBorreliagenospecies. MLSA was used to study genetic variations inBorreliastrains isolated fromI. ricinusticks collected from the woodlands in Skodje. The results demonstrate that the 50Borreliastrains were separated into 36 sequence types (STs) that were not previously represented in the MLST database. A distance matrix neighbour-joining tree (bootstrapped 500 iterations) showed four deeply branched clusters, and each deeply branched cluster represented oneBorreliagenospecies. The mean pairwise genetic differences confirm the genospecies clustering. The combination of alleles separates theBorreliastrains from northwest Norway from the strains in the MLST database, thus identifying new STs. Although a highly divergentB. afzeliipopulation could be expected, the heterogeneity among theB. gariniistrains is more unusual. The present study indicates that the circulation of strains between migrating birds and stationary birds in this coastal region may play a role in the evolution ofB. gariniistrains.

Prevalence and Diversity amongAnaplasma phagocytophilumStrains Originating fromIxodes ricinusTicks from Northwest Norway

The tick-borne pathogenAnaplasma phagocytophilumcauses great concern for livestock farmers. Tick-borne fever is a widespread disease in Norway, and antibodies have been produced amongst sheep, roe deer, red deer, and moose. The main vectorIxodes ricinusis found along the Norwegian coastline as far north as the Arctic Circle. A total number of 1804I. ricinusticks were collected and the prevalence of the pathogen was determined by species-specific qPCR. The overall infection rate varied from 2.83% to 3.32%, but there were no significant differences (p=0.01) in the overall infection rate in 2010, 2011, or 2012. A multilocus sequencing analysis was performed to further characterise the isolates. The genotyping of 27 strains resulted in classification into 19 different sequences types (ST), none of which was found in the MLST database. The nucleotide diversity was for every locus <0.01, and the number of SNPs was between 1 and 2.8 per 100 bp. The majority of SNPs were synonymous. A goeBURST analysis demonstrated that the strains from northwest Norway cluster together with other Norwegian strains in the MLST database and the strains that are included in this study constitute clonal complexes (CC) 9, 10, and 11 in addition to the singleton.