First Report of Chalara elegans on Roots of Black Elderberry

Elderberry (Sambucus nigra L.) is organically grown in Switzerland primarily for the flowers, which are used for the production of candies. In June of 2006 in a commercial orchard in Knonau, Switzerland, leaves prematurely turned yellow and dropped off. Other symptoms included weak growth of new twigs in the spring and wilting of leaves during the hotter temperatures of summer. Chlamydospores of Chalara elegans Nag Raj & Kendrick (synonym Thielaviopsis basicola (Berk. & Broome) Ferr.) (1) were found on fine roots of 4-year-old trees. In 2004, similar aboveground symptoms had been observed in two elderberry orchards in Emmental, Switzerland, but no causal organism was detected at that time. All three orchards were planted during 2002 with plants obtained from the same nursery. The pathogenicity of C. elegans on black elderberry was confirmed at the Agroscope Changins Wädenswil ACW Research Center in Conthey. Black elderberry seedlings were produced from seeds collected from a healthy plant in the fall of 2007. Seeds were placed on wet filter paper in petri dishes and incubated at 1°C for 6 months. Germinated seeds were transplanted in seed trays filled with steam-sterilized peat substrate and maintained in a greenhouse at 16 to 22°C. In the spring of 2008, a strain of C. elegans was recovered from carrot on potato dextrose agar (PDA) for artificial inoculation of elderberry. Mycelium edges of the isolate were transferred on two pieces of PDA to a clay/V8 medium. The medium consisted of 50 ml of expanded clay granules and 20 ml of clarified V8 juice in 250-ml Erlenmeyer flasks that had been autoclaved for 30 min. C. elegans cultures were incubated in darkness at 24°C for 1 month. Sandy loam soil with a pH of 7.4 (soil/water; 1:1 (wt/vol)) was autoclaved twice for 45 min with a 1-day interval. Plastic pots (0.5 liter) were first filled with 300 ml of sterile soil. Twenty milliliters of C. elegans inoculum were then added and covered with 100 ml of sterile soil. One black elderberry seedling with two true leaves fully expanded (3 to 5 cm high) was transplanted into each of five pots with inoculated clay granules and noninoculated clay granules. Plants were maintained in a greenhouse at 18 to 25°C and watered with tap water. Inoculated and control pots were kept in two separate trays to avoid cross contamination by drainage water. After 2 months, inoculated plants were approximately half the size of control plants. Approximately 75% of the inoculated roots were brown because of the occurrence of chlamydospores of C. elegans, which was reisolated on PDA. To my knowledge, this is the first report of C. elegans on roots of black elderberry. References: (1) K. H. Domsch et al. Compendium of Soil Fungi. Vol. 1. IHW-Verlag, Eching, Germany, 1993.

Molecular and Biological Characterization of a Mitovirus in Chalara elegans (Thielaviopsis basicola)

A 2.8-kb double-stranded RNA (dsRNA) element in strain BK18 of Chalara elegans originally isolated from cotton soil in California was characterized by obtaining a full-length cDNA sequence (2,896 nucleotides in length) from a series of overlapping clones. Sequence analysis revealed the presence of one large open reading frame (ORF I) using the mitochondrial genetic code, with 20 to 34% amino acid identity to the ORF I of other previously reported fungal mitochondrial RNA viruses. The ORF I encoded a putative protein of 705 amino acids and contained the conserved motif characteristic of RNA-dependent RNA polymerases. Purification of mitochondria from strain BK18 confirmed the co-localization of this dsRNA, and northern blot hybridization with a strand-specific probe revealed the (+) single-stranded nature. This Chalara elegans mitovirus (CeMV) is designated as a new member of the genus Mitovirus of the family Narnaviridae. Using dsRNA-specific primers, the ORF I region (positions 427 to 2544) was obtained from an additional 2.8-kb dsRNA element in strain HA2 originating from carrot roots in the Netherlands. Both ORFs had 98% homology at the nucleotide and amino acid levels. CeMV was also found to be present in five additional strains of C. elegans from different geographic locations worldwide, and a 97 to 100% nucleotide sequence identity was observed within a 300-bp region of ORF I in these strains. To determine the biological effects of CeMV on C. elegans, attempts to cure strain BK18 of the dsRNA were made. Sequential transfers of mycelium at 35 to 37°C yielded a colony which lacked the 2.8-kb dsRNA when visualized on agarose gels and also in northern blot hybridization analysis. However, reverse transcription-polymerase chain reaction with specific primer sets revealed a band, indicating that dsRNA replication had been significantly repressed (latent). The wild type and latently infected strains were compared for colony morphology, growth rate, melanin production, various enzymatic assays (polyphenoloxidase, laccase, tyrosinase, and esterase), and virulence on carrot roots. Colony morphology on V8 agar was comparable between the two strains, while growth rate, melanin production, and virulence were enhanced in the latently infected strain. There were no detectable differences in enzymatic activity. Transmission electron microscopy of hyphae of the wild type and latently infected strains revealed differences in the number and size of the mitochondria, which were enhanced in the latently infected strain. Our results show that CeMV is a new member of the genus Mitovirus with some disruptive effects on its fungal host and is present in C. elegans strains from different locations worldwide.

Effect of the Nicotiana debneyi black root rot resistance gene on the yield and quality characteristics of flue-cured tobacco in Ontario

The effects of Nicotiana debneyi-derived resistance to black root rot disease were evaluated for yield, agronomic and quality traits by comparing the near isogenic cultivars AC Gayed (resistant) and Delgold (susceptible). Over 7 yr of trials the possession of resistance led to yields and economic returns that averaged 6 and 7% lower, respectively, than for the susceptible line. Key words: Flue-cured tobacco, Nicotiana tabaccum, Black Root Rot, Chalara elegans, Nicotiana debneyi, yield, quality

Morphological and molecular characterization of Chalara elegans (Thielaviopsis basicola), cause of black root rot on diverse plant species

The extent of variation in colony morphology and chlamydospore size, septation, and pigmentation was studied in 50 isolates of Chalara elegans Nag Raj et Kendrick (syn. Thielaviopsis basicola (Berk. et Br.) Ferr.) originating from 12 different geographic areas and substrates. In addition, the extent of genetic variation among these isolates was determined using random amplified polymorphic DNA (RAPD) analysis. Five general morphological groups could be distinguished among the isolates, two of which were aberrant phenotypes (albino and mycelial) that were derived upon continuous subculture of some wild-type isolates in the laboratory. The isolates with the most variation in phenotype originated from British Columbia and California. Six primers (10-mers) were used to generate 90 bands in RAPD-PCR, of which 75 were polymorphic. A high degree of diversity was apparent within C. elegans, and some banding patterns generated by specific primers were unique to certain isolates, thereby generating fingerprints. Distinct groups (clusters) were obtained following UPGMA analysis and, generally, these were composed of isolates from similar geographic regions or hosts. However, isolates from some areas, for example, British Columbia, were also found to belong to different clusters. There was generally a good relationship between groups assigned on the basis of morphology and those derived from cluster analysis, that is, isolates within a cluster tended to have similar morphology. In a few isolates, the aberrant phenotypes (albino and mycelial) could be distinguished using RAPDs from the wild type by the absence of 1 or 2 bands, indicating that changes in the nucleotide sequence had occurred, possibly through mutation. The average similarity index among all 50 isolates of C. elegans was 87%. An outgroup species (Chalara thielaviodes) had a similarity value of 40%.