First report of root rot on Manglietia decidua caused by Calonectria ilicicola in China

Manglietia decidua (Magnoliaceae) was a class I endangered plant in China. During 2018-2020, a severe root rot (about 10% - 90% disease incidence ) was observed on 2-year-old seedlings in the nursery in Yichun, Jiangxi province (N27°52’20”; E114°27’46”). Symptoms started on leaves showing dehydration and chlorosis, the root of diseased plant became black and rotted, and in severe cases, the plants withered and died. The symptomatic root tissues were cut and dipped in a 3% hydrogen peroxide solution for 5 mins, rinsed thrice with sterile water, and then placed on potato glucose agar medium containing ampicillin (50mg/L). The plates were kept in an incubator at 25-28°C for 2-3 days in the dark. The Calonectria-like fungus was consistently isolated from 100% of tissues and the colonies were feathery, moderate white aerial mycelium, surface pale brown, reverse with white outer margin, and brown inner region. The perithecia produced on carnation leaf agar were solitary, subglobose to ovoid, dark red-brown, and measured 273.8 - 427.2 × 362.6 - 628.9 µm (av. 360.9 × 429.9 µm) (n = 31). Clavate asci contained eight spores and tapered into a long thin stalk. Ascospores were hyaline, guttulate, straight to slightly curved with rounded ends, 36.8- 66.1×4.4-7.3 μm (av. 49.9 × 5.9 μm) (n = 52), 1-septate, constricted at the septum and aggregated in the upper third of the ascus. On PDA，conidia formed on penicillate conidiophores within 10 days were hyaline, 1(-3)-septate, cylindrical, rounded at both ends, straight, 36.5-61.7 × 5.0-7.2 μm (av. 50.7 × 6.2 µm) (n=48). Isolate HML 20 and 27 were used to further confirm species identity by five loci analysis：ITS (MZ389092 and MZ389093), ACT (MZ398252 and MZ398253), HIS3 (MZ398254 and MZ398255), TEF1-α (MZ398256 and MZ398257), and TUB2 (MZ398258 and MZ398259). NCBI BLASTN showed the high sequence identity with Calonectria ilicicola ex-type culture CBS 190.50 (CMW 30998) (Liu et al 2020): 100 % for ITS (MT359727), TUB2 (AY725631), and HIS3 (MT335506), 99.22% for ACT (MT335036), 99.80% for TEF1-α (MT412797). Maximum likelihood (ML) analysis and Bayesian inference (BI) based on the combined ITS, tub2, his3 and tef1 sequence using RAxML v.1.0.0 and MrBayes v. 3.2.1 software revealed that isolate HML 20 and 27 clustered together with C. ilicicola strains in C. kyotensis species complex. Thus, the fungus was identified as C. ilicicola (anamorph: Cylindrocladium parasiticum) based on morpho-molecular criteria (Lombard et al. 2010). Pathogenicity was determined under greenhouse conditions (25-30 ℃). The 2-year-old plants grown in 25-cm pots for 20 days were inoculated. Five 6-mm mycelial plugs from 7-day culture on PDA were buried 5 cm under the soil adjacent to the unwounded taproot of each plant and the plants were watered regularly to keep the soil moisture content at about 15%. After ten days, inoculated plants began to show chlorosis symptoms on leaves and collapsed within 15 to 20 days, while no symptoms were observed on control plants. The same colonial fungus was successfully reisolated. Calonectria ilicicola is an economically important plant pathogen worldwide, which causes diseases on Arachis hypogaea, Cinnamomum kanahirai, Glycine max, Medicago sativa, Sassafras randaiense, and Vaccinium spp. etc. in China (Gai et al 2017, Fei et al 2018, Zhang et al 2020 ). As far as we know, it is first report of C. ilicicola causing root rot on M. decidua. At present, this disease is an important threat to the conservation of M. decidua.

Survey of potential root pathogens in softwood cuttings collected from Georgia blueberry nurseries

Survey sampling of 18 blueberry nurseries propagating softwood cuttings was conducted in 2007 and 2008 to determine which soilborne plant pathogens were most prevalent in commercial blueberry propagation systems in southern Georgia. Samples were collected four times: June (early), September (mid), and October (late) in the 2007 growing season, with additional sampling of overwintered cuttings in April 2008. This survey revealed that Calonectria ilicicola (Cylindrocladium parasiticum), causing Cylindrocladium root rot, is a primary pathogen of blueberry cuttings in southern Georgia. The organism was isolated consistently during all four survey dates with a cutting-level incidence of 3.6, 10.2, 36.4, and 14.3% in the first through fourth samplings, respectively. Rhizoctonia spp. were recovered less frequently and were only present during the first (10.9% cutting-level incidence), third (4.6%), and fourth survey dates (3.6%), while the Oomycetes Pythium and Phytophthora were detected only on the second survey date. Fusarium spp. were isolated commonly (29.1% incidence in the first, 12.2% in the second, 18.2% in the third, and 7.1% in the fourth sampling), but pathogenicity remains uncertain. Across all survey dates, Calonectria, Rhizoctonia, Oomycetes, and Fusarium were recovered at least once from 41.2, 17.6, 0.1, and 82.4% of nurseries surveyed, respectively. When nursery-level pathogen presence-absence data collected from this survey was analyzed in relation to a survey of production practices used by these same propagators, the practice of reusing growth media was found significantly associated with Calonectria presence.

The Whole Genome Sequence Resource of Calonectria ilicicola, the Casual Pathogen of Soybean Red Crown Rot

Calonectria ilicicola (ana. Cylindrocladium parasiticum) is a soilborne plant pathogenic fungus with a broad host range, and it can cause red crown rot of soybean and Cylindrocladium black rot of peanut, which has become an emerging threat to crop production worldwide. Limited molecular studies have focused on Calonectria ilicicola and one of the possible difficulties is the lack of genomic resource. This study presents the first high quality and near-completed genome of C. ilicicola using the Oxford Nanopore GridION sequencing platform. A total of 16 contigs were assembled and the genome of C. ilicicola isolate F018 was estimated to have 11 chromosomes. Currently, the C. ilicicola F018 genome represents the most contiguous assembly, which has the lowest contig number and the highest contig N50 among all Calonectria genome resources. Putative protein-coding sequences and secretory proteins were estimated to be 17,308 and 1,930 in the C. ilicicola F018 genome, respectively; and the prediction was close to other plant pathogenic fungi such as Fusarium species within the Nectriaceae family. The availability of this high-quality genome resource is expected to facilitate research on fungal biology and genetics of C. ilicicola, and to support the understanding on pathogen virulence and disease management.

Evaluation of Glycine max and Glycine soja for Resistance to Calonectria ilicicola

Breeding for resistance to soybean red crown rot (Calonectria ilicicola) has long been hampered by the lack of genetic sources of adequate levels of resistance to use as parents. Mini core collections of soybean (Glycine max) originating from Japan (79 accessions), from around the world (80 accessions), and a collection of wild soybeans (Glycine soja) consisting 54 accessions were evaluated for resistance to C. ilicicola (isolate UH2-1). In the first two sets, average disease severity scores of 4.2 ± 0.28 and 4.6 ± 0.31 on a rating scale from zero for no symptom to 5.0 for seedling death were recorded from the set from Japan and the world. No high levels of resistance were observed in these two sets. On the other hand, disease severity score of 3.8 ± 0.35 for the wild soybean accessions was somewhat lower and exhibited higher levels of resistance compared to the soybean cultivars. Three accessions in the wild soybean collection (Gs-7, Gs-9, and Gs-27) had disease severity score ≤2.5 and showed >70% reduction in fungal growth in the roots compared to soybean control cv. “Enrei”. Further analysis using 10 C. ilicicola isolates revealed that accession Gs-9 overall had a wide range of resistance to all isolates tested, with 37% to 93% reduction in fungal growth relative to the cv. Enrei. These highly resistant wild soybean lines may serve as valuable genetic resources for developing C. ilicicola-resistant soybean cultivars.

Development of Three Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Calonectria ilicicola, Dactylonectria macrodidyma, and the Dactylonectria Genus in Avocado Roots

Black root rot of avocado is a severe disease of nursery trees and young orchard transplants, causing tree death within a year after planting. In Australia, key pathogens include species complexes Calonectria ilicicola and Dactylonectria macrodidyma; however, several other Dactylonectria species also cause the disease. Rapid detection of these pathogens in planta is important to speed up implementation of disease management and reduce loss. The purpose of this study was to develop three loop-mediated isothermal amplification (LAMP) diagnostic assays to rapidly identify species within the C. ilicicola and D. macrodidyma complexes and species in the Dactylonectria genus in avocado roots. Primers were designed from β-tubulin sequence data of C. ilicicola and from histone H3 of D. macrodidyma and the Dactylonectria genus. The LAMP primers were tested for specificity and sensitivity with 82 fungal isolates, which included the target species complexes C. ilicicola and D. macrodidyma; species within the target Dactylonectria genus viz. D. macrodidyma, D. anthuriicola, D. novozelandica, D. pauciseptata, and D. vitis; and isolates of nontarget species, including Calonectria sp., Cylindrocladiella sp., Gliocladiopsis forsbergii, G. peggii, G. whileyi, Ilyonectria sp., Mariannaea sp., Fusarium sp., and Phytophthora cinnamomi. The species-specific LAMP assays were sensitive and specific at DNA concentrations of 1 pg/µl for C. ilicicola and 0.01 ng/µl for D. macrodidyma, whereas the Dactylonectria genus-wide assay was sensitive to 0.1 ng/µl. Detection of C. ilicicola occurred within 10 to 15 or 15 to 30 min when the template was pure DNA or crude extracts obtained from suspending fungal cultures in sterile water, respectively. Detection of D. macrodidyma was between 12 to 29 min with pure DNA and 16 to 30 min with crude extracts. Dactylonectria spp. were detected within 6 to 25 min with pure DNA and 7 to 23 min with crude extracts. The specificity of the assays was found to be dependent on time and isothermal amplification temperature, with optimal specificity occurring in reactions of <30 min and at temperatures of 67°C for C. ilicicola and D. macrodidyma assays and 69°C for Dactylonectria genus-wide assays. The assays were modified to accommodate a DNA extraction step and use of avocado roots as DNA templates. Detection in avocado roots ranged between 12 to 25 min for C. ilicicola, 12 to 26 min for D. macrodidyma, and 14 to 30 min for species in the Dactylonectria genus. The LAMP assays are applicable across multiple agricultural industries, because C. ilicicola, D. macrodidyma, and Dactylonectria spp. are also important pathogens of various crops and ornamental plants.