Boxwood (Buxus sp. L.) is a very popular evergreen shrub in the United States which is widely used as landscape plant and fresh greenery. Boxwood 'Green velvet' (B. sinica var. insularis x B. sempervirens) plants grown in field condition exhibiting Volutella blight symptoms were found in a commercial nursery in Warren Co., Tennessee in May 2019. Leaves appeared red, brown or tan color on affected plants. Waxy, salmon pink colored fruiting bodies (sporodochia) were observed underneath the affected leaves using a hand lens (Figure 1). Leaf drop was also observed on plants. Black lesions under the bark were observed in some of the plants. The disease severity (percentage leaf area diseased) was nearly 40% and the disease incidence was nearly 30% of 1,000 plants. Infected leaf and stem tissues collected from four symptomatic plants were surface sterilized with 70% ethanol and washed with sterile distilled water. Culturing the infected leaf and stem pieces, 5-mm in size, on potato dextrose agar (PDA) consistently yielded white fluffy aerial mycelium growth with scattered salmon-color slimy masses of conidia forming from sporodochia after 10 days incubation at 25°C in a 12-h fluorescent light and dark cycle. A total of two isolates (FBG2020_396 and FBG2020_405) were hyphal tip purified on PDA. The conidia (n = 50) were hyaline, aseptate, fusiform to ellipsoidal measuring average of 7.8 × 3.3 μm (range: 4.84 to 13.2 μm × 2.2 to 4.64 μm). To confirm the pathogen identity, total DNA was extracted using UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) directly from a 5-day old culture of isolates (FBG2020_396 and FBG2020_405) on PDA. The ribosomal DNA internal transcribed spacer region (ITS), β-tubulin (tub2) and part of 28S large ribosomal subunit (LSU) regions were amplified by PCR using the primer pairs ITS 5/ITS 4, T1/BTb2 and LR0R/LR5, respectively (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; Rehner and Samuels 1994; Vilgalys and Hester 1990; White et al. 1990). Newly generated sequences - GenBank/NCBI acc. nos. MW459251, MW465902 (ITS), MW464656, MW464657 (tub2) and MW459255, MW465903 (LSU) were 100% identical to Pseudonectria foliicola L. Lombard & Crous ex-type (CBS 123190) sequences KM231776, KM232035 and NG_058095, respectively. To complete Koch's postulates, six boxwood 'Green velvet' plants grown in 10 cm square pots (containing 40% coarse sand and 60% ground pine bark) were inoculated by spraying conidial suspension of P. foliicola [FBG2020_396 (1 × 105 conidia/mL)] obtained from 2-wk-old PDA cultures. Plants were covered with clear plastic humidity domes for 3 days and then they were maintained in a growth chamber at 25°C and 60% RH in a 12-h fluorescent light and dark cycle. Six control boxwood plants were maintained in the same environment without pathogen introduction. Pathogenicity test was conducted twice. After 10 days, typical symptoms of Volutella blight developed on the inoculated plants and microscopic examination revealed the same pathogen morphology as the original isolate. Pseudonectria foliicola was consistently re-isolated from leaves and stems. All control boxwood plants remained symptom-free and P. foliicola was not isolated from the leaves or stems. Pseudonectria foliicola causing Volutella blight has been reported on B. sempervirens in Czech Republic (Spetik et al. 2020), New Zealand (Lombard et al. 2015); Buxus sp. in Illinois, Maryland, Massachusetts, North Carolina and Washington (Salgado-Salazar et al. 2019). To our knowledge, this is the first report of Volutella blight of boxwood caused by P. foliicola in Tennessee. Pseudonectria foliicola is an opportunistic pathogen and infects weak, stressed, and injured boxwood plants/cuttings (Rivera et al. 2018). This pathogen could cause a serious economic loss to boxwood nursery growers, as it can significantly affect the ornamental value of boxwood plants and fresh greenery. Integration of sanitation practices with other disease management strategies such as biorational products and reduced-risk fungicides will be necessary for limiting the spread of pathogen and successful management of P. foliicola on boxwood in both field and postharvest conditions.
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