First Report of Leaf Spot Caused by Corynespora cassiicola on Viburnum odoratissimum Ker-Gawl. var. awabuki (K. Koch) Zabel ex Rumpl. (sweet viburnum) in China

Sweet viburnum [Viburnum odoratissimum Ker-Gawl. var. awabuki (K. Koch) Zabel ex Rumpl.] belonging to the family Adoxaceae, is a medical and landscape plant, native to Korea (Jeju Island), Taiwan, and Japan (Edita 1988). In June and September 2019, leaf spots were observed on approximately 65% to 80% of sweet viburnum plants in a hedgerow located in Fenghe Xincheng District (28°41'52.9"N 115°52'14.3"E) in Nanchang, China. Initial symptoms of disease appeared as dark brown spots surrounded by red halos (Figure 1 A), which expanded irregularly. Finally, the center of the lesions desiccated and became light-brown, surrounded by a deep-red halos (Figure 1 B). Ten leaf samples with typical symptoms were collected and washed with tap water for about 15 min. The tissue between the healthy and necrotic area (ca. 4 mm × 4 mm) was cut with a sterile scalpel and surface sterilized with 70% alcohol for 45 s, 2% NaClO for 2 min, washed in sterile deionized water three times, dried on sterilized filter paper, then placed in Petri dishes and incubated at 25℃ in the dark. After 3 to 5 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA dishes. Eventually, 54 fungal isolates were obtained and, of these, 39 isolates were identical in their morphological characteristics. Morphological analysis was performed according with Ellis (1971). The isolate S18, chosen as representative, formed a gray to grayish brown colony with concentric circleson PDA, and a diameter of 8.5 to 9 cm after 7 days incubation at 25℃ (Figure 1 G). Conidia were hyaline, straight or slightly curved, needle shaped, truncate at the base, and acuminate at the tip, with 2 to 6 pseudosepta, 18.90 to 38.38 µm (avg. = 27.51 µm) × 1.64 to 4.50 µm (avg. = 2.60 µm) (n = 36) (Figure 1 H). The genes of fungal isolates (i.e., ITS, tub2 and ACT) were amplified with ITS4/ITS5 for ITS (White, Bruns et al. 1990), Bt2a/Bt2b for tub2 (Glass and Donaldson 1995) and ACT783R/ACT512F for ACT (Carbone and Kohn 1999) and sequenced. The sequences were deposited in GenBank (MW165772 for ITS, MW175900 for ACT and MW168659 for tub2), which showing greater than 99.1% similarity to multiple C. cassiicola accessions, respectively. Pathogenicity tests were performed on healthy leaves in field by inoculating surface-sterilized mature leaves with puncture wound (Figure C) and non-wounded young leaves with 20 µL of a conidial suspension (105 conidia ml-1) (Figure F and G) at 26℃. After 4 to 7 days, all inoculated leaves reproduced similar symptoms as observed initially in the field (Figure 1 C, E and F). To fulfill Koch’s postulates, the fungus was isolated on PDA from the margins of leaf spots on inoculated leaves and confirmed as C. cassiicola by morphological characters and ITS gene sequencing. Previously, C. cassiicola was reported as an endophyte on Viburnum spp. and Viburnum odoratissimum (Alfieri et al. 1994). More recently, C. cassiicola has been reported as a pathogen of many plant species in China, such as kiwifruit (Cui, Gong et al. 2015), American sweetgum (Mao, Zheng et al. 2021), castor bean (Tang, Liu et al. 2020), and holly mangrove (Xie, He et al. 2020). To our knowledge, this is the first report of leaf spot disease on sweet viburnum caused by C. cassiicola in China and the precise identification of the causal agent will be useful for its management.

First Report of Alternaria spp. Causing Leaf Spot on Sweet Viburnum in China

Sweet viburnum [Viburnum odoratissimum (L.) Ker Gawl] is an evergreen shrub mainly cultivated along roadsides in urban landscapes and also in parks and residential areas. A foliar disease occurred on about 40% of sweet viburnum plants near Anhui Grand Theatre, Anhui Province of China in June 2019. In early stages of sweet viburnum infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. The spots developed on the upper, middle, and lower leaves of the plant, however, the upper leaves got more severely affected. As the disease develops, the spots enlarged and became rectangular or oval, brown to dark-brown, and their centers became ashen gray. In later stages of infection, the diseased leaves became wilting. Diseased leaves were surface disinfested and three small sections (2-3 mm2) were cut from the margin of the lesions. Sections were placed in 1.5% NaClO for 2 min, submerged in three changes of sterilized distilled water for 1 min each, placed onto potato dextrose agar (PDA) medium amended with 50 μg/ml of ampicillin and kanamycin, and incubated at 25℃ for 3 days. The mycelium from the leading edge of colonies growing from the tissue was sub-cultured onto a PDA plate for 3 days, followed by spore induction (Simmons 2007) and single spore isolation to obtain a pure culture of the putative pathogen. Colonies of one single spore isolate HF0719 were rounded, grayish white with dense aerial mycelium viewed from above and dark brown viewed from below. On potato carrot agar (PCA) medium, conidiophores were branched or occasionally unbranched. On branched conidiophores, conidia were in dwarf tree-like branched chains of 2-5 conidia. On unbranched conidiophores, conidia were simple or in chains of 2-8 conidia. Conidia were light brown or dark brown, ovoid, ellipsoidal to fusiform, and ranged in size from 7 to 26.5 × 4.5 to 11 μm with an average size of 16 × 7 µm based on 500 spore observations, with one beak and 1-7 transverse, 0-3 longitudinal, and 0-3 oblique septa. Beaks were ranged in (1.5-)2-10(-16) μm long. Based on cultural and morphological characteristics, isolate HF0719 was identified as Alternaria spp. (Simmons 2007). For molecular identification, total genomic DNA was isolated from mycelia collected from 7 day-old colonies of isolate HF0719 using the fungal genomic DNA extraction kit (Solarbio, Beijing, China). Fragments of five genes, including those encoding glyceraldehyde-3-phosphate dehydrogenase (gpd), plasma membrane ATPase, actin, calmodulin, and the Alternaria major allergen (Alt a1) regions of isolate HF0719 were amplified and sequenced using primer pairs gpd1/gpd2 (Berbee et al. 1999), ATPDF1/ATPDR1, ACTDF1/ACTDR1, CALDF1/CALDR1 (Lawrence et al. 2013), and Alt-for/Alt-rev (Hong et al. 2005), respectively. The obtained nucleotide sequences were deposited into GenBank as accession numbers: gpd, MT614365; ATPase, MT614364; actin, MT614363; calmodulin, MN706159; and Alt a1, MN304720. Phylogenetic tree using a maximum likelihood bootstrapping method based on the five-gene combined dataset in the following order: gpd, ATPase, actin, calmodulin, Alt a1 of HF0719 and standard strains representing 120 Alternaria species (Lawrence et al. 2013) was constructed. Isolate HF0719 formed a separate branch. On the basis of morphological characteristics and phylogenetic pattern, isolate HF0719 was identified as Alternaria spp.. A pathogenicity test was performed by rubbing 32 healthy leaves of six 5-year-old sweet viburnum plants with a cotton swab dipped in spore suspension containing 2.6 × 106 spores/ml, following leaf surface disinfection with 70% ethanol in the open field. Sterilized distilled water was used as control. The average air temperature was about 28℃ during the period of pathogenicity test. Eleven days after inoculation, 100% of inoculated leaves showed the leaf spot symptom identical to symptoms observed in the field. Control leaves were symptomless. The experiment was done three times. The re-isolated pathogen from the leaf lesion had the same morphological and molecular characteristics as isolate HF0719, thus satisfying Koch’s postulates. The genus Alternaria has been reported to cause leaf spot on sweet viburnum in Florida, USA (Alfieri et al. 1984). To our knowledge, this is the first report of Alternaria spp. causing leaf spot on sweet viburnum in China, a highly valued ornamental plant. Our findings will contribute to monitoring and adopting strategies for manage leaf spot disease on sweet viburnum.

Nitrogen Fertilizer Rate, Timing, and Application Method Affects Growth of Sweet Viburnum and Nitrogen Leaching from Simulated Planting Beds

Several Florida cities and counties ban fertilization during the summer rainy season (fertilizer blackout). Little research is available to support or contradict the underlying justifications for these policies. We used large-volume lysimeters to address the impacts of several fertilization regimes on plant growth and aesthetics of sweet viburnum (Viburnum odoratissimum Ker Gawl.) and nitrogen (N) leaching from landscape beds during shrub establishment and maintenance. Three levels of N fertilization (98, 195, and 293 kg·ha−1), two levels of application method (per plant and broadcast), two levels of fertilization timing (regular and blackout), and an unfertilized control (0 kg·ha−1 N) were applied to lysimeters in a completely randomized design with three replicates (3 × 2 × 2 factorial plus untreated control). Increasing fertilization rate increased plant growth and improved plant quality, but also increased N leaching from lysimeters. Including a summer fertilization blackout period reduced nitrate + nitrite (NO3 + NO2-N) loading from lysimeters during sweet viburnum establishment [0 to 28 weeks after planting (WAP)] compared with year-round fertilization at the same total N rate without adversely impacting plant growth or aesthetics. However, NO3 + NO2-N loads from lysimeters were higher when fertilizers were applied on the summer blackout application schedule during the shrub maintenance period. Targeted (per plant) fertilization beneath the dripline of sweet viburnum at an annual N rate of 195 kg·ha−1 can maintain plant health while limiting N leaching losses on a year-round or blackout fertilization schedule.

Growth and Quality Response of Woody Shrubs to Nitrogen Fertilization Rates during Landscape Establishment in Florida

Despite inconsistent reports of nitrogen (N) fertilization response on growth of landscape-grown woody ornamentals, broad N fertilization recommendations exist in the literature. The objective of this research was to evaluate the growth and quality response of three landscape-grown woody shrub species to N fertilizer. Three ornamental shrub species, ‘Alba’ indian hawthorn (Raphiolepis indica), sweet viburnum (Viburnum odoratissimum), and ‘RADrazz’ (Knock Out™) rose (Rosa) were transplanted into field soils in central Florida (U.S. Department of Agriculture hardiness zone 9a). Controlled-release N fertilizer was applied at an annual N rate of 0, 2, 4, 6, and 12 lb/1000 ft2 for 100 weeks. Plant size index measurements, SPAD readings (a measure of greenness), and visual quality ratings were completed every month through 52 weeks after planting (WAP) and then every 3 months through 100 WAP. Plant tissue total Kjeldahl N (TKN) concentrations and shoot biomass were measured at 100 WAP. Results of regression analysis indicated little to no plant response (size index, biomass, SPAD) to N fertilizer rate. Shrub quality was acceptable for all species through 76 WAP regardless of the N fertilization rate. However, quality of rose and sweet viburnum fertilized with N at the low rates (<2 lb/1000 ft2) was less than acceptable (<3 out of 5) after 76 WAP. Results suggest that posttransplant applications of fertilizer may not increase plant growth, but that low-to-moderate levels of N fertilization (2 to 4 lb/1000 ft2 per year) may help plant maintain quality postestablishment.

Posttransplant Irrigation Frequency Affects Growth of Container-grown Sweet Viburnum in Three Hardiness Zones

The survival and quality of shrubs planted in the landscape from containers is dependent on irrigation to ensure the development of a healthy root system. This study determined the effect of irrigation frequency on survival, quality, canopy growth index, root to canopy spread ratio, and dry root and shoot biomass of Viburnum odoratissimum Ker-Gawl. (sweet viburnum) planted in Florida in USDA hardiness Zones 8b (Citra, FL), 9a (Balm, FL), and 10b (Ft. Lauderdale, FL). Sweet viburnum shrubs were planted into the landscape from 11.4-L (#3) containers and irrigated with 3 L every 2, 4, or 8 days. Shrubs were planted on eight dates over a 2-year period (2004 to 2006). Irrigation frequency during the 12- to 22-week irrigation period had no significant effect on sweet viburnum survival or aesthetic quality at any location. In addition, there was no irrigation effect on root spread, root to shoot biomass ratio, or root biomass for shrubs planted in Zones 8b or 9a. However, sweet viburnum irrigated every 2 days had greater canopy growth index at 28 and 104 weeks after planting than shrubs irrigated every 4 or 8 days in Zone 8b and every 8 days in Zone 9a. When planted in Zone 10b, sweet viburnum irrigated every 2 days exhibited greater growth index, shoot biomass, and root biomass than plant receiving irrigation every 4 days. Although more frequent irrigation (every 2 days) resulted in more plant growth in Zones 8b and 10b, sweet viburnum survived and grew after planting under natural rainfall conditions provided they were irrigated with 3 L of water every 8 days during establishment until roots reached the canopy edge in hardiness Zones 8b and 9a and every 4 days in hardiness Zone 10b. Subsequent supplemental irrigation (hand-watering) was only needed after irrigation was ended when plants exhibited visible signs of drought stress and there was no measurable rainfall for 30 consecutive days.

Consequences of Excessive Overhead Irrigation on Runoff during Container Production of Sweet Viburnum

The effects of irrigation rate on volume and nutrient content of runoff were investigated. Runoff (leachate plus un-intercepted irrigation and rain) was collected weekly for 20 weeks during production of trade #1 (2.7-liter) sweet viburnum [Viburnum odoratissimum (L.) Ker-Gawl.] fertilized with a resin-coated, controlled-release fertilizer [Osmocote 18N–2.6P–10K (18–6–12), 8–9 month 21C (70F)]. Treatments were a factorial arrangement of two irrigation rates [1 (IRR1) or 2 (IRR2) cm/day (0.39 or 0.79 in)] and two fertilizer rates [15 (FRT15) or 30 (FRT30) g/container (0.53 or 1.06 oz)]. Total runoff volume was 970 liters/m2 (2380 gal/100 ft2) for IRR1 and 2220 liters/m2 (5450 gal/100 ft2) for IRR2 which was 49 and 69%, respectively, of total irrigation plus rainfall. Increasing the irrigation rate from 1 to 2 cm/day increased leaching losses of N, P, and K 34, 38, and 45%, respectively, with FRT15 and 21, 28, and 23%, respectively, with FRT30. Increasing the irrigation rate increased nutrient loads (g/m2) but decreased nutrient concentrations (mg/liter) in runoff.

Effects of Container Spacing Practice and Fertilizer Placement on Runoff from Overhead-Irrigated Sweet Viburnum

Information on how management practices affect runoff volume and nutrient content is needed to improve irrigation and fertilizer efficiency while minimizing environmental impacts. Runoff (leachate plus unintercepted irrigation and rain) was collected weekly for 20 weeks during production of sweet viburnum (Viburnum odoratissimum (L.) Ker-Gawl.) in trade #1 (2.7 liter) containers fertilized with 15 g (0.53 oz) of a resin-coated, controlled-release fertilizer Osmocote 18N–2.6P–10K (18–6–12), 8–9 month 21C (70F)] and overhead-irrigated with water at 1 cm/day (0.39 in). Treatments were a factorial arrangement of two container spacing practices [spaced at planting (SP) or spaced midseason (SM)] and two fertilizer placement methods [incorporated (INC) or surface-applied (SURF)]. Cumulative runoff volume averaged 1590 liters/m2 (3900 gal/100 ft2) or 66% of irrigation plus rain and was 9% higher for SP than SM. A 37% reduction in shoot dry weight of SP versus SM plants was attributed to heat stress in SP containers. SURF decreased N, P, and K leaching losses (mg/container) 42, 42, and 25%, respectively, at SP and 16, 25, and 4%, respectively, at SM. Nutrient leaching losses as a percent of applied were 11–18% for N, 7–13% for P, and 19–28% for K. Total nutrient loads in runoff were 4.6–11.1 g/m2 for N, 0.48–1.25 g/m2 for P, and 5.8–10.1 g/m2 for K with peak nutrient loss occurring during the first two weeks after planting.

Irrigation Affects Landscape Establishment of Burford Holly, Pittosporum, and Sweet Viburnum

Ilex cornuta Lindl. & Paxt. ‘Burfordii Nana’ (dwarf burford holly), Pittosporum tobira [Dryand]. ‘Variegata’ (pittosporum), and Viburnum odorotissimum Ker Gawl. (sweet viburnum) were transplanted into field plots in an open-sided, clear polyethylene-covered shelter to evaluate growth, aesthetic quality, and establishment rates in response to 2-, 4-, or 7-d irrigation frequencies. Establishment was delayed 1 to 2 months for I. cornuta ‘Burrford Nana’ irrigated every 7 d compared with 2- and 4-d frequencies; however, growth and aesthetic quality were similar among treatments. Plants irrigated every 7 d also had higher cumulative water stress levels. Leaf area, shoot dry weight, and total biomass increased among P. tobira ‘Variegata’ and V. odorotissimum irrigated every 2 d. Pittosporum tobira ‘Variegata’ and V. odorotissimum irrigated every 2 d also had greater canopy size and root dry weight, respectively. Neither cumulative water stress nor establishment was affected by irrigation frequency for either species.