Margaret T. Mmbaga

Identification of Alternaria alternata as a Causal Agent for Leaf Blight in Syringa Species

While many isolates of Alternaria alternata are common saprophytes on trees and shrubs, this study clearly demonstrated that A. alternata is a primary pathogen in lilac (Syringa sp.), causing a leaf-blight that affects different Syringa species. Isolates of Alternaria sp. were collected from leaf blight samples of lilacs in the field. The internal transcribed spacer (ITS) region and morphological characterization were used to identify lilac blight pathogen. Based on 100% ITS nucleotide sequence identities to the Alternaria genus in the GenBank and morphological features, these isolates were identified as A. alternata. Disease symptoms were reproduced in lilac plants inoculated with A. alternata mycelial plugs and sprayed with a fungus-free culture filtrate, indicating that pathogenesis in lilac involves secondary metabolites or toxins. Diagnostic primers were developed to detect Alternaria sp. and A. alternata in lilac leaf blight based on ITS region and four known genes associated with pathogenesis in A. alternata: mixed-linked glucanase precursor, endopolygalacturonase, hsp70, and histone genes. The results from our study indicated A. alternata is a primary pathogen in lilac leaf blight, and these diagnostic primers can be used as a tool for the fast detection of A. alternata associated with lilac leaf blight.

Powdery Mildew of Dogwoods: Current Status and Future Prospects

Cornus is a large genus of trees and shrubs that are collectively referred to as dogwoods. Flowering dogwood (C. florida L.) and kousa dogwood (C. kousa (F. Buerger ex Miq.) Hance) and interspecific hybrids of these species are popular ornamental trees that are known for their showy bracts, red berries (drupes), and/or fall color. Other species that are commercially grown for specialty markets include the pagoda dogwood (C. alternifolia L.), giant dogwood (C. controversa Hemsl.), cornelian cherry (C. mas L.), Pacific dogwood (C. nuttallii Aud.), and redosier dogwood (C. sericea L.). The foliage of native species, such as flowering dogwood and pagoda dogwood, is high in calcium (12) in quantities above what is needed for skeletal growth of wildlife; it is the preferred browse material for lactating does in late spring while many other trees are still leafless (13,22). The berries of flowering dogwood have high oil content and provide mast for numerous species of migrant songbirds, wild turkeys, and large and small mammals (22). For many years, nurseries that produced flowering and kousa dogwoods had the luxury of working with relatively diseasefree crops. Disease management and control costs were minimal and estimated at approximately

IN VITRO REGENERATION OF THE TENNESSEE CONEFLOWER (ECHINACEA TENNESSEENSIS)

120/ha/year. In the late 1970s, flowering and kousa dogwoods were threatened by a new disease, dogwood anthracnose, caused by Discula destructiva (39), which was reviewed by Daughtrey et al. (3). In 1994, another disease, powdery mildew, reached epiphytotic levels in flowering dogwoods. Tens of millions of dollar’s worth of dogwoods were destroyed and millions of cultivated seedlings lost their commercial value because formal management strategies were not formulated. In subsequent years, fungicide management costs were estimated to be

Winter Survival and Source of Primary Inoculum of Powdery Mildew of Dogwood in Tennessee

1,975/ha/year. Many small producers of dogwoods terminated production of the tree because they could not afford the additional overhead or were not inclined to continue routine fungicide sprays every 2 weeks from May to October. Powdery mildew on C. florida was first reported in 1887 by Burrill and Earle (1), but this disease was rarely reported on flowering dogwood in the United States before 1994. However, the disease appeared simultaneously in forest, landscape, and nursery plantings statewide in Alabama in 1994 (8). Similar outbreaks of powdery mildew were observed in Tennessee, where many nursery fields of flowering dogwood were abandoned (Fig. 1). Powdery mildew has emerged as a nationwide disease of flowering dogwood (2). Although the host side of the disease triangle remained constant, we do not know whether the change of frequency and severity of powdery mildew in flowering dogwood was due to a change in the pathogen or a change in the environment.

Perpetuation of Powdery Mildew Infection and Identification of Erysiphe australiana as the Crape Myrtle Pathogen in Mid-Tennessee

SummaryTennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.

First report of Myrothecium roridum causing leaf spot on garden hydrangea in the United States.

A 3-year study (1996-1998) on the epidemiology of dogwood powdery mildew showed that Microsphaera pulchra is the primary powdery mildew pathogen of dogwoods (Cornus spp.) in mid-Tennessee, and the occurrence of Phyllactinia guttata is insignificant. Cleistothecia harvested from leaf debris in spring contained viable asci and ascospores and produced powdery mildew infection on disease-free plants. Ascospores that were morphologically similar to those of M. pulchra were trapped on sticky slides in the vicinity of dogwoods throughout spring. Previously infected plants that did not harbor cleistothecia failed to develop signs of infection under growth-chamber and greenhouse conditions conducive to powdery mildew infections. Results from these studies indicate that cleistothecia on leaf debris rather than mycelia on dormant buds constitute the main winter survival structure and primary sources of spring inoculum in mid-Tennessee.

PCR-based markers for detection of Colletotrichum acutatum and C. gloeosporioides in flowering dogwood (Cornus florida)

The fungus Erysiphe lagerstroemiae is commonly known as the powdery mildew pathogen in crape myrtle (Lagerstroemiae indica) in the United States, and Erysiphe australiana is the powdery mildew pathogen reported in Japan, China, and Australia. The teleomorph often used to identify powdery mildew fungi rarely develops in crape myrtle, and in our observations, ascocarps never formed. Our study showed that the crape myrtle pathogen overwintered as mycelia on dormant buds. The internal transcribed spacer (ITS) regions of rDNA and the intervening 5.8S rRNA gene were amplified using standard polymerase chain reaction (PCR) protocols and the universal primer pairs ITS1 and ITS4. PCR products were analyzed by electrophoresis in a 1.5% agarose gel and sequenced, and the ITS PCR product was 666 bp from ITS1/ITS4 and 704 bp from ITS1-F/ITS4. BLAST analysis of the sequence of the PCR products showed identical similarity with E. australiana reported in Japan, China, and Australia. Comparison of ITS sequences with information in the GenBank on other powdery mildew fungi showed a closest alignment (93% similarity) to Erysiphe juglandis that infects walnut. Specific primers for E. australiana were developed and evaluated for use as diagnostic tools. Out of 12 specific primer pairs evaluated, four primer pairs and four double primer pairs were highly specific to E. australiana and did not amplify Erysiphe pulchra of dogwood, Erysiphe syringae of common lilac, Erysiphe circinata of maple, or Phyllactinia guttata of oak. The E. australiana-specific primers amplified 16 samples of crape myrtle powdery mildew collected from diverse locations in mid-Tennessee. These results clearly showed that the crape myrtle powdery mildew in mid-Tennessee was caused by E. australiana. Specific primers reported in this article provide a diagnostic tool and may be used to confirm the identity of crape myrtle powdery mildew pathogen in other areas in the United States and wherever the disease occurs.

Estimation of narrow sense heritability of powdery mildew resistance in pseudo F2 (F1) population of flowering dogwoods (cornus Florida L)

Garden hydrangea (Hydrangea macrophylla) is a popular flowering shrub that grows well in Tennessee but foliar diseases impact their appearance, health, and market value. Leaves of garden hydrangea showed necrotic lesions with concentric rings of brown and dark brown at the Tennessee State University Research Center in McMinnville. A fungus was recovered from June and July leaf samples with 20% frequency of isolation from approximately 40 leaf pieces that were surface sterilized and plated in potato dextrose agar (PDA). Isolates developed white colonies and dark gray-to-black, spore-bearing mycelial cushions (sporodochia) that formed on older colonies (30 to 45 days old) at 25 ± 2°C. Conidia were hyaline to slightly dark, one-celled, ovoid to elongate with rounded ends, and 2.0 to 2.5 × 5.5 to 6.5 μm. These morphological characteristics were consistent with those described for Myrothecium roridum Tode ex Fr. (1). DNA sequence for three isolates of this fungus showed identical internal transcribed spacer (ITS) region sequences (GenBank Accession No. HM215150) with 99% maximum sequence identity to M. roridum isolates (GenBank Accession Nos. AJ301994.1 and AJ608978). Another close match (97%) was with M. gramineum (GenBank Accession No. FJ235084) and M. tongaense (GenBank Accession No. AY254157). Pathogenicity of M. roridum was evaluated on detached leaves from three hydrangea cultivars, Nikko Blue, All Summer Beauty, and Blue bird. Four, medium-size, detached leaves were placed in moist chambers and inoculated with 5-mm mycelial plugs from 14-day-old cultures; sterile PDA was used as the control treatment. A randomized, complete-block experimental design was used with a replication of four leaves per cultivar. Incubation temperature was 26 ± 2°C. Necrotic lesions started 4 to 5 days after inoculation in all inoculated leaves; lesions expanded to cover 10 to 25% of the leaf surface and formed concentric rings; sterile PDA plugs did not produce leaf lesions. This experiment was repeated twice and similar symptoms were produced; M. roridum was reisolated from all inoculated leaves. Spray inoculation of detached leaves of hydrangea cv. Pretty Maiden with 5 × 104 spores/ml produced similar symptoms; leaves sprayed with water remained symptom free. M. roridum has a wide host range and similar symptoms have been reported on other ornamentals including salvia (2), begonia ( http://mrec.ifas.ufl.edu/foliage/folnotes/begonias.htm ), gardenia ( http://cfextension.ifas.ufl.edu/agriculture/ nursery_production/ documents/Gardenia.pdf ), and cotton (3). To our knowledge, this is the first report of M. roridum causing leaf spot on H. macrophylla in the United States. References: (1) M. B. Ellis. Page 465 in: More Damatacous Hyphomycetes. CABI, Wallingford, UK. 1993. (2) J. A. Mangandi et al. Plant Dis. 91:772, 2007. (3) R. L. Munjal. Indian Phytopathol. New Delhi, 13:150, 1960.

Cherry leaf spot disease management in ornamental cherries in mid-Tennessee

Two fungi, Colletotrichum acutatum (=Glomerella acutata) and C. gloeosporioides (=G. cingulata) were isolated from flowering dogwood (Cornus florida) and characterised by molecular PCR-based markers. The internal transcribed spacer (ITS) universal primer pair ITS1-F/ITS4 produced fragments of 622 bp and 613 bp in C. acutatum and C. gloeosporioides, respectively. The two sequences were 91.1% identical in a 626 bp region. Six ITS specific primer pairs were designed that produced PCR fragments only for C. acutatum. The 18S small subunit rRNA universal primer pair NS1/NS2 produced fragments of 570 bp and 560 bp for C. acutatum and C. gloeosporioides, respectively. The two fragments were 97.4% identical in a 497 bp region. Two specific primer pairs were designed that produced PCR fragments only for the laccase or pectate lyase B gene of C. gloeosporioides. Two additional primer pairs were designed that produced specific PCR fragments only for the glutamine synthetase or a key lime pathogenicity gene of C. acutatum. These results provide useful tools to identify and distinguish C. acutatum and C. gloeosporioides in limb dieback of flowering dogwood, and this research also provides an approach for further characterisation of pathogenicity related genes.

Interaction of bean leaf pubescence with rust urediniospore deposition and subsequent infection density

Powdery mildew disease caused by Erysiphe (sect. Microsphaera) pulchra a (Cook & Peck, Braun & Takamatsu) is a devastating disease in flowering dogwoods (Cornus Florida) throughout southeastern USA where flowering dogwood is widely grown. Host resistance is the most sustainable and economical way for controlling this disease, but resistance breeding has been slow and genetic information on powdery mildew resistance is unavailable. In this study, pseudo F2 populations derived from crosses between selected resistant (R) and susceptible (S) plants (RxR, RxS and SxR), were evaluated for disease severity at two locations in three years, McMinnville in 2005 and 2012, and McMinnville and Nashville, TN, in 2013. Disease reaction in progeny from reciprocal crosses was similar indicating that there was no cytoplasmic inheritance of resistance genes. Disease severity of progenies showed continuous distributions, suggesting that powdery mildew resistance is inherited quantitatively. High narrow sense heritability (0.74 in 2005, 0.84 in 2012 and 0.90 in 2013) and broad sense heritability (0.9) in parental clones were observed. This information will be useful in breeding programs for dogwood improvement.