The Rlm13 Gene, a New Player of Brassica napus–Leptosphaeria maculans Interaction Maps on Chromosome C03 in Canola

Canola exhibits an extensive genetic variation for resistance to blackleg disease, caused by the fungal pathogen Leptosphaeria maculans. Despite the identification of several Avr effectors and R (race-specific) genes, specific interactions between Avr-R genes are not yet fully understood in the Brassica napus–L. maculans pathosystem. In this study, we investigated the genetic basis of resistance in an F2:3 population derived from Australian canola varieties CB-Telfer (Rlm4)/ATR-Cobbler (Rlm4) using a single-spore isolate of L. maculans, PHW1223. A genetic linkage map of the CB-Telfer/ATR-Cobbler population was constructed using 7,932 genotyping-by-sequencing-based DArTseq markers and subsequently utilized for linkage and haplotype analyses. Genetic linkage between DArTseq markers and resistance to PHW1223 isolate was also validated using the B. napus 60K Illumina Infinium array. Our results revealed that a major locus for resistance, designated as Rlm13, maps on chromosome C03. To date, no R gene for resistance to blackleg has been reported on the C subgenome in B. napus. Twenty-four candidate R genes were predicted to reside within the quantitative trait locus (QTL) region. We further resequenced both the parental lines of the mapping population (CB-Telfer and ATR-Cobbler, > 80 × coverage) and identified several structural sequence variants in the form of single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and presence/absence variations (PAVs) near Rlm13. Comparative mapping revealed that Rlm13 is located within the homoeologous A03/C03 region in ancestral karyotype block “R” of Brassicaceae. Our results provide a “target” for further understanding the Avr–Rlm13 gene interaction as well as a valuable tool for increasing resistance to blackleg in canola germplasm.

First Report on Colletotrichum siamense Causing Anthracnose of Castor Bean in Zhanjiang, China

Castor bean (Ricinus communis L.) is an important oil crop. Anthracnose lesions were observed on leaves of castor bean at the stage of budding and fruiting in field (21˚17'51''N, 110˚18'16''E), Zhanjiang, Guangdong Province, China in August 2019. The incidence rate was approximately 40% (n=600 investigated plants). Early symptoms were yellow spots appearing from the edge or the tip of the leaves. Later, the spots gradually expanded and became dark brown, which coalesced into larger irregular or circular lesions (Supplementary Figure 1). Seven diseased leaves were collected from seven plants. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A single-spore isolate (RLC-1) was used for further study. The colony of isolate RLC-1 on PDA was white to gray in color with cottony mycelia in 6 days at 28 °C. Conidia were one-celled, hyaline, cylindrical, clavate, obtuse at both ends and measured 14.2 to 18.5 µm × 3.8 to 5.5 µm (n =50). Appressoria were oval to irregular in shape, dark brown, and ranged from 7.3 to 10.5 µm × 5.7 to 6.5 µm (n = 20). Morphological characteristics of isolate RLC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate RLC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), and ACT (ACT-512F/ACT-783R) (Weir et al. 2012). Analysis of the ITS (accession no. MN880199), GAPDH (MN884048), and ACT (MN891766) sequences revealed a 99%–100% identity with the corresponding ITS (JX010250), GAPDH (KX578786), ACT (JX009541) sequences of C. siamense in GenBank. A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that clustered the isolate RLC-1 with C. siamense with the type strain ICMP 19118 (Supplementary Figure 2). Morphological characteristic and phylogenetic analysis identified the isolate RLC-1 associated with anthracnose of castor bean as C. siamense. Pathogenicity test was performed in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Twenty healthy plants of Zi Bi No. 5 castor bean (2 month old) were grown in pots with one plant in each pot. Inoculation was conducted on leaves with mycelial plugs of RLC-1 or agar plugs (as control). Three plugs were considered for each leaflet. Ten plants were used in each treatment (five for wounded inoculation and five for unwound inoculation). Anthracnose lesions as earlier were observed on the leaves after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all inoculated leaves was identical to the isolate RLC-1 by morphology and ITS analysis but not from control plants. C. siamense causes anthracnose on various plant hosts, including mango in Colombia (Pardo-De la Hoz et al. 2016) and Rosa chinensis in China (Feng et al. 2019) but not including castor bean. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on castor bean. Thus, this work provides a basis for focusing on the management of the disease in future.

Whole genome sequence of an edible and potential medicinal fungus, Cordyceps guangdongensis

ABSTRACTCordyceps guangdongensis is an edible fungus which has been approved as a Novel Food by the Chinese Ministry of Public Health in 2013. It also has a broad application prospect in pharmaceutical industries with many medicinal activities. In this study, the whole genome of C. guangdongensis GD15, a single spore isolate from a wild strain, was sequenced and assembled with Illumina and PacBio sequencing technology. The generated genome is 29.05 Mb in size, comprising 9 scaffolds with an average GC content of 57.01%. It is predicted to contain a total of 9150 protein-coding genes. Sequence identification and comparative analysis indicated that the assembled scaffolds contained two complete chromosomes and four single-end chromosomes, showing a high level assembly. Gene annotation revealed a diversity of transporters that could contribute to the genome size and evolution. Besides, approximately 15.49% and 13.70% genes involved in metabolic processes were annotated by KEGG and COG respectively. Genes belonging to CAZymes accounted for a proportion of 2.84% of the total genes. In addition, 435 transcription factors (TFs) were identified, which were involved in various biological processes. Among the identified TFs, the fungal transcription regulatory proteins (18.39%) and fungal-specific TFs (19.77%) represented the two largest classes of TFs. These data provided a much needed genomic resource for studying C. guangdongensis, laying a solid foundation for further genetic and biological studies, especially for elucidating the genome evolution and exploring the regulatory mechanism of fruiting body development.

Permanent Draft Genome Sequence forFrankiasp.Strain EI5c, a Single-Spore Isolate of a Nitrogen-Fixing Actinobacterium, Isolated from the Root Nodules ofElaeagnus angustifolia

Frankiasp. strain EI5c is a member ofFrankialineage III, which is able to reinfect plants of theEleagnaceae,Rhamnaceae,Myricaceae, andGymnostoma, as well as the genusAlnus. Here, we report the 6.6-Mbp draft genome sequence ofFrankiasp. strain EI5c with a G+C content of 72.14 % and 5,458 candidate protein-encoding genes.

First Report of Anthracnose on Pepper Fruit Caused by Colletotrichum scovillei in Brazil

Anthracnose is major disease of pepper (Capsicum annum) in the tropics and causes severe damage both in the field and postharvest. In Brazil, this disease is caused by Colletotrichum acutatum, C. boninense, C. capsici, C. coccodes, and C. gloeosporioides, where the first species is responsible for 70% of all occurrences (3). Recently, C. acutatum has been considered a species complex (1); thus, the aim of this study was to verify the etiology of anthracnose on peppers using a morphological and molecular approaches. In 2011, pepper fruits with typical symptoms of anthracnose (dark, sunken spots with concentric rings of orange conidial masses) were collected in Viçosa, Minas Gerais, Brazil. A single spore isolate was obtained on potato dextrose agar (PDA), and the derived culture was deposited in the Coleção de Culturas de Fungos Fitopatogênicos “Prof. Maria Menezes” (code CMM-4200). The upper side colonies on PDA were gray, cotton-like, and pale gray to pale orange. Conidia were hyaline, aseptate, smooth, straight, cylindrical with round ends or occasionally with end ± acute, 12.5 to 17 μm long and 3.5 to 4 μm wide on synthetic nutrient deficient agar. The isolate was morphologically typical of species belonging to the C. acutatum complex. Molecular identification of the pathogen was carried out and sequences of the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (βt) were obtained and deposited in GenBank (Accession Nos. KJ541821 to KJ541823). A search in the Q-bank fungi database using the ITS, βt, and GAPDH sequences retrieved C. scovillei with 100% identity for all three genes. This pathogen was previously reported in Capsicum spp. only in Thailand, Indonesia, and Japan (1,2). To confirm pathogenicity, drops with 105 spores/ml were deposited in 10 artificially wounded fruits (cv. Itapuã 501 and Melina). In control fruits, drops of sterilized water were deposited onto wounds. The fruits were covered for one day with a transparent plastic bag with moisture supplied by a wet filter paper. The fruits were detached and mature. The bags were removed, and the fruits were incubated for 10 days in a growth chamber at 25°C with a photoperiod of 12 h. After 4 days, gray-brown to black sunken spots with concentric rings were observed on 100% of the wounded fruits that had been inoculated. No disease was observed on the control fruits. The fungus C. scovillei was successfully re-isolated from symptomatic fruits to fulfill Koch's postulates. To our knowledge, this is the first report of anthracnose on pepper fruit caused by C. scovillei in Brazil. Due to the diversity of species that cause anthracnose in Capsicum, future studies using morphological and molecular tools are essential for the correct identification of Colletotrichum spp. on pepper in Brazil. References: (1) U. Damm et al. Stud. Mycol. 73:37, 2012. (2) T. Kanto et al. J. Gen. Plant. Pathol. 80:73, 2014. (3) M. J. Z. Pereira et al. Hortic. Bras. 29:569, 2011.

First Report of Myrothecium roridum Causing Myrothecium Leaf Spot on Dieffenbachia picta ‘Camilla’ in Taiwan

Dumb cane (Dieffenbachia picta (Lodd.) Schott ‘Camilla’), family Araceae, is a popular houseplant in Taiwan. During the winter of 2012, dumb canes with dark brown concentric spots on leaves and bright yellow borders were found in a protected ornamental nursery in Wandan township, Pingtung County, Taiwan. On diseased leaves, fungal fruiting bodies were sometimes observed in the concentric lesions and a fungal isolate was consistently isolated from the lesions. A single spore isolate, myr 2-2, was maintained on potato dextrose agar (PDA) for further tests. To fulfill Koch's postulates, the spores of myr 2-2 were suspended in sterilized distilled water containing 0.05% of Tween 20, 1 × 105 conidia ml–1, and then sprayed on leaves of D. picta ‘Camilla’ growing in polypropylene plant pots (about 7 cm in diameter), three plants per treatment. For the control, three plants were sprayed with sterilized distilled water containing 0.05% of Tween 20. Both inoculated and non-inoculated plants were covered with plastic bags and incubated in a growth chamber at 26 ± 1°C. Nine to 12 days after inoculation, symptoms described above were observed on inoculated plants whereas the plants in control remained healthy. The same fungus was reisolated from inoculated plants but not from the controls. Furthermore, the fungal pathogen was identified using its physiological, morphological, and molecular characteristics. In the mycelial growth test, the diameter of the fungal colony reaches 58.2 mm on PDA at 25°C after 14 days. The colonies were floccose, white to buff, and sporulate in concentric zones with olivaceous black to black sporodochia bearing viscid masses of conidia. Conidia were narrowly ellipsoid with rounded ends. The average size of 100 conidia was 6.25 ± 0.04 × 1.63 ± 0.02 μm. For molecular identification, the rDNA internal transcribed spacer (ITS) of isolate myr 2-2 was PCR amplified using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3′) primer pairs (3) and sequenced. The rDNA sequence was deposited in GenBank (KC469695) and showed 100% identity to the Myrothecium roridum isolates BBA 71015 (AJ302001) and BBA 67679 (AJ301995) (4). According to the physiological, morphological (1,2), and molecular characteristics, the fungal isolate was identified as M. roridum Tode ex Fr. To the best of our knowledge, this is the first report of Myrothecium leaf spot caused by M. roridum on D. picta ‘Camilla’ in Taiwan. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , January 31, 2013. (2) M. Tulloch. Mycol. Pap. 130: 1-42, 1972. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990. (4) Y. X. Zhang et al. Plant Dis. 95:1030, 2011.

Reaction of Lines of the Rapid Cycling Brassica Collection and Arabidopsis thaliana to Four Pathotypes of Plasmodiophora brassicae

The clubroot reaction of five Rapid Cycling Brassica Collection (RCBC) lines (Brassica carinata, B. juncea, B. napus, B. oleracea, and B. rapa) and 84 lines of Arabidopsis thaliana to pathotypes 2, 3, 5, and 6 of Plasmodiophora brassicae (as classified on William's system) was assessed. Also, the reaction of the Arabidopsis lines to a single-spore isolate of each of pathotypes 3 and 6 was compared with that of a field isolate. Seedlings were inoculated with resting spores of P. brassicae, maintained at 25 and 20°C (day and night, respectively), and assessed for clubroot incidence and severity at 6 weeks after inoculation. Several lines of A. thaliana and RCBC exhibited a differential response to pathotype but none of the lines were immune. Among the RCBC lines, B. napus was resistant to all of the pathotypes; B. oleracea was resistant to pathotypes 2, 3, and 5; B. carinata and B. rapa were resistant to pathotypes 2 and 5; and B. juncea was susceptible to pathotypes 5 and 6 and had an intermediate response to pathotypes 2 and 3. Line Ct-1 of A. thaliana was highly resistant to pathotype 2, Pu2-23 was highly resistant to pathotype 5, and Ws-2 and Sorbo were highly resistant to pathotype 6. These results indicate that the lines of RCBC and A. thaliana have potential for use as model crops for a wide range of studies on clubroot, and could be used to differentiate these four pathotypes of P. brassicae. The reaction of the RCBC lines to pathotype 6 was highly correlated with response under field conditions but the reaction to the single-spore isolates of pathotypes 3 and 6 was not strongly correlated with reaction to the field collections in the Arabidopsis lines.

First Report of Fludioxonil Resistance in Botrytis cinerea from a Strawberry Field in Virginia

Gray mold caused by Botrytis cinerea Pers.:Fr. is one of the most economically important diseases of cultivated strawberry (Fragaria × ananassa) worldwide. Control of gray mold mainly depends on fungicides, including the phenylpyrrole fludioxonil, which is currently marketed in combination with cyprodinil as Switch 62.5WG (Syngenta Crop Protection, Research Triangle Park, Raleigh, NC). In 2012, 790 strains of B. cinerea were collected from 76 strawberry fields in eight states, including Arkansas, Florida, Georgia, Kansas, Maryland, North Carolina, South Carolina, and Virginia. Strains were collected from sporulating flowers and fruit and sensitivity to fludioxonil was determined using a conidial germination assay as previously described (2). Only one isolate from a farm located in Westmoreland County, Virginia, grew on medium amended with the discriminatory dose of 0.1 μg/ml fludioxonil and was therefore considered low resistant. The isolate did not grow on 10 μg/ml. All other 789 isolates did not grow at either of the two doses. This assay was repeated twice with a single-spore culture of the same strain. In both cases, residual growth was observed on the fludioxonil-amended medium of 0.1 μg/ml. The single spore isolate was confirmed to be B. cinerea Pers. using cultural and molecular tools as described previously (1). To assess resistance in vivo, commercially grown ripe strawberry fruit were rinsed with sterile water, dried, placed into plastic boxes (eight strawberries per box for each of the three replicates per treatment), and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of fludioxonil (Scholar SC, Syngenta) to runoff using a hand mister. This dose reflects the rate recommended for gray mold control according to the Scholar label. Each fruit was stabbed at three equidistant points, each about 1 cm apart and 1 cm deep using a syringe tip. Wounds were injected with a 30-μl droplet of conidia suspension (106 spores/ml) of either 5 sensitive or the resistant isolate. Control fruit were inoculated with water. After inoculation, the fruit were kept at 22°C for 4 days. In two independent experiments, sensitive and low resistant isolates were indistinguishable in pathogenicity on detached, unsprayed fruit. The low resistant isolate developed gray mold disease on all treated and untreated fruit (100% disease incidence) as determined by the absence or presence of gray mold symptoms. The sensitive isolates only developed disease on untreated fruit. The EC50 values, determined in microtiter assays with concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μg/ml fludioxonil, were 0.01 μg/ml for the sensitive isolates and 0.26 μg/ml for the resistant isolate. To our knowledge, this is the first report of fludioxonil resistance in B. cinerea from strawberry in North America. Our monitoring results indicate that resistance is emerging 10 years after the introduction of fludioxonil and stress the importance of chemical rotation for gray mold control. References: (1) X. P. Li et al. Plant Dis. 96:1634, 2012. (2) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

Adaptation to Brassica Host Genotypes by a Single-Spore Isolate and Population of Plasmodiophora brassicae (Clubroot)

Plasmodiophora brassicae, the cause of clubroot of crucifers, is an increasingly important pathogen of canola (Brassica napus) in Alberta, Canada. In response, clubroot-resistant canola genotypes are being deployed to help reduce yield losses. Two experiments were conducted to examine the effect on P. brassicae virulence of repeated exposure of a population and single-spore isolate of the pathogen to the same host. The first experiment examined changes in the index of disease over five cycles of infection on seven Brassica hosts (European Clubroot Differential [ECD] 02, ECD 04, ECD 05, ECD 15, ‘45H26’, ‘45H29’, and 08N823R). The second experiment tested the virulence of five cycled populations (‘45H29’, 08N823R, ECD 05, and ECD 15) and three cycled single-spore isolates (‘45H29’, 08N823R, and ECD05) on four resistant canola genotypes (‘73-77’, ‘73-67’, VT-SD-09, and ‘9558C’). The results from these experiments clearly demonstrate the ability of both single-spore isolates and populations of P. brassicae to rapidly erode the resistance present in the two canola genotypes, ‘45H29’ and 08N823R. Although the index of disease increased on these two genotypes, the four resistant canola genotypes remained resistant to all the cycled populations and single-spore isolates in the second experiment. These results underscore the importance of crop rotation in the management of clubroot in Alberta.