Investigation of Fungi Causing Twig Blight Diseases on Peach Trees in South Carolina

A survey of fungal pathogens causing twig blight on commercial peach trees was conducted in South Carolina in the fall of 2016. Shoots with cankers, pycnidia, and dieback were collected from six locations around the state. Isolates obtained from these samples were identified as Botryosphaeria obtusa, Phomopsis amygdali, Leucostoma persoonii, and Cytospora sp., based on colony morphology, conidia size and shape, and ribosomal DNA sequence analysis. L. persoonii was the most prevalent species and was isolated in five of the six locations, followed by P. amygdali and B. obtusa. The sensitivity of representative isolates of B. obtusa, P. amygdali, and L. persoonii to fungicides of different FRAC codes was evaluated. All species tested were sensitive to thiophanate-methyl (FRAC 1) and pyraclostrobin and azoxystrobin (both FRAC 11), whereas all species were resistant to boscalid and fluopyram (both FRAC 7). P. amygdali and B. obtusa were sensitive to difenoconazole and propiconazole (both FRAC 3), whereas L. persoonii was moderately resistant. L. persoonii was the most virulent species based on expansion of mycelium in the cambium layer of 2-year-old, detached twig pieces. Bacterial spot (BS)-sensitive cultivar ‘O’Henry’ was most susceptible to B. obtusa compared with BS-sensitive ‘Summerprince’, brown rot (BR)-resistant ‘Contender’, and BR-sensitive ‘Coronet’ but was least susceptible to L. persoonii. Coronet was most susceptible to L. persoonii. There were no significant differences between susceptibility of the cultivars to P. amygdali. This study established that L. persoonii is currently the most frequent twig blight pathogen in South Carolina, perhaps owing to its superior fitness. Some fungicides were effective in controlling all twig blight pathogens and may therefore be useful for chemical management strategies. Our study also provides the first evidence that the genetic basis of resistance to BS and BR in peach trees is not necessarily linked to tolerance to wood pathogens.

Effect of Different Growing Systems of Apple on Trunk and Branch Diseases and Pests

The presented study aimed to determine apples trunk and branch diseases and pests in three growing systems conventional, integrated and biological (organic). The investigations were made on an experimental apple orchard (1 ha) of the Institute of Agriculture at Kyustendil, Southwest Bulgaria in four consecutive years from 2007 to 2010. Three scab resistant cultivars Prima, Florina and Erwin Baur grafted on rootstocks MM106 were planted in 1996. The orchard was divided into four plots. One plot was treated conventionally with a normal pesticide programme, two plots were treated integrated according to the general principles, rules and standards of integrated apple production and one plot for biological (organic). The monitoring of pests and diseases and assessment of their density were done every two weeks. It was established that during the experimental period important disease and pests on apple trees in different growing systems were black rot Botryosphaeria obtusa, apple clearwig moth Synanthedon myopaeformis and shorthole borer Scolytus rugulosus. The damages by trunk and branch diseases and pests on apple were considerable higher in biological growing system. The mean rate of attack of cultivar Erwin Baur by Botryosphaeria obtusa in biological and conventional growing systems was 52.35% and 4.65%, respectively. The percentage of damaged by Scolytus rugulosus trunk and branch area per tree reach to 58.74 in biological and 0.23 in conventional system. Reduced vitality of apple trees growing with out pesticides and mineral fertilizers in biological growing system was the reason for strong infection of Botryosphaeria obtusa and attack of Synanthedon myopaeformis and Scolytus rugulosus.

First Report of Botryosphaeria obtusa as Causal Agent of Olive Tree Branch Dieback in Tunisia

A branch dieback of olive trees (Olea europaea L. cv. Manzanilla de Sevilla) was observed in 2010 in an orchard (50 ha), located in the Testour region of northern Tunisia. More than 50% of trees were severely damaged by the disease. Symptomatic trees presented dead branches and wilted leaves, which remained attached to the shoots, and the affected tissues appeared abnormally dark compared with the inner bark of healthy branches. Numerous pycnidia were observed on the surface of the infected branches. For diagnosis, symptomatic stems were collected and small pieces of discolored tissues were excised from lesion margins, surface sterilized in 0.5% sodium hypochlorite for 1 min, rinsed and dried on sterilized filter paper, then placed on acidified Difco potato dextrose agar plates (APDA; 2.5 ml of 25% lactic acid per liter). Plates were incubated at 25°C for 4 to 5 days, and hyphal tips from developing fungal colonies were transferred to PDA and placed under fluorescent light (12 h/day). A fastgrowing, pycnidia-producing fungus was consistently isolated, with conidia exuding onto the agar surface of 10-day-old cultures. On the basis of colony characteristics, isolates were identified as Botryosphaeria obtusa (3). Conidia were large, dark brown, aseptate, rounded at both ends or truncate at base, and 25 to 26.8 × 10.5 to 12.03 μm. Pathogenicity tests were performed on detached stems of cv. Manzanilla by 7-mm diameter mycelial plugs cut from actively growing cultures of the fungus. Stems (30 cm length) were cleaned, surface sterilized with sodium hypochlorite (0.25% for 2 min), and wounded with a sterilized scalpel. Mycelial disks were placed over wounds and wrapped with Parafilm to prevent desiccation. Control stems were mock inoculated with sterile agar plugs. Inoculated and control stems were placed in polyethylene boxes and incubated at 25°C. After 45 days, inoculated stems developed brown discoloration, and small dark pycnidia appeared on stem surfaces. Controls remained healthy. Koch's postulates were verified by isolating the fungus from symptomatic stems. To confirm the identification, DNA of one isolate was extracted and the fungal primers ITS1 and ITS4 (4) were used to amplify the internal transcribed spacer region of rDNA. Purified amplicons were sequenced and a BLAST search of the GenBank database revealed 99% homology with B. obtusa isolate HO166525.1. The anamorph of the fungus, Diplodia seriata, has been recognized as the cause of fruit rot of olive (1) and branch canker or dieback (2). To our knowledge, this is the first report of a canker disease of olive trees caused by B. obtusa in Tunisia. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. Taylor et al. Australas. Plant Pathol. 34:187, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Grapevine Trunk Disease Caused by Botryosphaeria obtusa in China

In September 2010, grapevine (Vitis vinifera) trunk diseases were observed in several vineyards of Yantai District in Shandong Provinces and Changli County of Hebei Provinces of China. Characteristic symptoms of Botryosphaeria canker were apparent, including dark brown discoloration on the trunk (visible in cross-section), cob base shriveling, drying of fruit clusters, and berry falling (2). To identify the causal pathogen, culturing of fungi was attempted from 387 small pieces of tissue from the canker margins of 43 diseased plants. Samples were surface disinfected by placing them in 75% ethanol for 1 min and rinsing with sterilized water three times before culturing on potato dextrose agar (PDA) at 28°C for 7 to 10 days. Fungi isolated were single spored to obtain pure cultures. On the basis of colony characteristics on PDA, 18 isolates from the 387 tissue pieces were eventually identified as Botryosphaeria obtusa (1), Most of the other fungi isolated were B. dothidea. B. obtusa colonies were grayish white, becoming dark brown with age, and pycnidia were formed after incubation for approximately 7 days. Conidia measured 8 to 11 × 17 to 26 μm (n= 50). Two isolates were used for rDNA internal transcribed spacer (ITS) sequence analysis with primers ITS1 and ITS4 (3). PCR products were separated by electrophoresis and bands were purified for legation with PMD-18T (Takara Company, Dalian, China) vector for sequencing. BLAST searches of two ITS sequences had 99 to 100% identity to B. obtusa. EF1-α and β-tubulin sequence analysis gave similar results. Koch's postulates were completed in the greenhouse on grape shoots inoculated with two isolates of B. obtusa originally isolated from diseased plants in the field. Inoculations were made on green shoots of V. vinifera cv. Dunkelfelder T. Six shoots were inoculated per isolate by wounding with a 4-mm cork borer (2 mm deep) and placing a colonized agar plug from a 5-day-old culture on the wound and wrapping it with Parafilm. Controls were mock inoculated with an agar plug from sterile PDA. Inoculated shoots were incubated in the dark under moist conditions in the laboratory for 8 to 10 days at 25°C. Inoculated shoots had necrotic cankers after 8 to 10 days and B. obtusa was recovered from each canker margin. The results indicated that some grapevines in China with symptoms of Botryosphaeria canker were infected by B. obtusa. To our knowledge, this is the first report of this pathogen causing trunk disease on grapevine in China. References: (1) A. Taylor et al. Australas. Plant Pathol. 34:187, 2005. (2) J. R. Úrbez-Torres et al. Plant Dis. 92:519, 2008. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Parasitic and saprophytic fungi on the species in the genus Paulownia

The species in the genus Paulownia are distributed in Asia. In Serbia, the most represented species is P. tomentosa, widely used as an ornamental species in town green spaces. Other species - P. fortunei and P. elongata are also cultivated in forest plantations. The aim of this paper is to present the most frequent parasitic and saprophytic fungi on the species in the genus Paulownia and to point out their significance. Phyllactinia guttata, Ascochyta paulowniae and Phyllosticta paulowniae were described on the leaves and Diaporthe eres and Botryosphaeria obtusa were described on the bark.

Production of Botryosphaeria species conidia using grapevine green shoots

Methods for producing large numbers of conidia were developed to allow investigation of the pathogenicity of five Botryosphaeria species (B lutea B australis B obtusa B parva and B stevensii) previously isolated from symptomatic grapevines Pycnidium formation of all five species was low on five mycological media tested Further any pycnidia produced on the media contained few conidia An alternative method was developed whereby grapevine green shoots were infected with Botryosphaeria species the lesions air dried and then induced to sporulate under moist conditions Botryosphaeria australis B lutea and B stevensii produced abundant pycnidia oozing conidia within 36 h The air dried stem pieces could still produce pycnidia that oozed conidia after 3 months of storage at room temperature Botryosphaeria parva produced few pycnidia that did not release any conidia Botryosphaeria obtusa lesions were small and restricted to the inoculation point with only a few pycnidia and conidia being produced

First Report of Diplodia seriata Causing Shoot Blight and Cankers of Cotoneaster salicifolius in Bulgaria

Shoot and branch blight was observed on shrubs of Cotoneaster salicifolius in the region of Plovdiv, Bulgaria in autumn and winter surveys during 2005 and 2006. Scattered pycnida were found on the withered branches, which bore sunken cankers with marginal bark cracks. Fungal colonies obtained from affected host tissue developed on potato dextrose agar (PDA) at 24 to 25°C in the dark. Colonies grew rapidly, were floccose, initially white, becoming grayish brown, and finally went from gray to black. Dark brown, unilocular pycnida formed after 2 to 3 weeks in culture. Conidia that formed at the tips of cylindrical, percurrently, proliferating, conidiogenous cells were ovoid, straight, pale brown to dark brown, internally verruculose, aseptate or occasionally with a single median septum. The mean conidial dimensions were 20 × 12 μm, the extreme range was 15 to 25 × 11 to 15 μm. These characters correspond to Diplodia seriata De Not., which is the anamorph of “Botryosphaeria” obtusa (2). Identity was confirmed from the nucleotide sequences of the internal transcribed spacer (ITS) from the rRNA repeat as described elsewhere (1). BLAST searches in GenBank showed 100% identity of isolates with reference sequences of D. seriata including that from the ex-epitype culture. The ITS sequence of a representative isolate (CAP337) has been deposited in GenBank (Accession No. EU483658). Pathogenicity was tested by inserting 3-mm-diameter mycelia plugs from 7-day-old PDA cultures into scalpel-wounded mature apple fruits (cv. Granny Smith) and wounded shoots of C. salicifolius. Sterile PDA plugs were placed into similarly made wounds of control specimens. There were three replicates for each treatment and the inoculated wounds were subsequently covered with Parafilm. Small necrotic lesions occurred on the apple fruits 3 to 5 days after inoculation and after 10 to 12 days on the shoots. Within 4 to 5 weeks, all apple fruits were entirely rotten, shrunken, and covered with pycnida, whereas the shoots withered and typical blight symptoms developed above the inoculation sites. The pathogen was reisolated from all inoculated samples but not from any control treatment. To our knowledge, this is the first report of D. seriata on C. salicifolius in Bulgaria. It is likely that this pathogen will be found increasingly more frequently because of the intensive introduction of decorative Rosaceae species into urban districts. References: (1) A. Alves et al. Mycologia 96:598, 2004. (2) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.

First Report of Diplodia seriata, the Anamorph of “Botryosphaeria” obtusa, Causing Fruit Rot of Olive in Spain

In October, 2006, 1,000 olive fruits (Olea europaea) were collected to study latent infections of Colletotrichum spp., causal agent of anthracnose, in an olive orchard in Córdoba Province (southern Spain). The fruits were incubated at 22 to 24°C and 100% relative humidity. Thirteen percent of the fruits showed a black rot that covered part or all of the fruit and pycnidia formed on the fruit surface. The fungus most consistently isolated from affected fruits was identified as Diplodia seriata, the anamorph of “Botryosphaeria” obtusa, by morphological criteria (1). Pathogenicity tests were performed on 50 immature and 50 mature fruits of olive cv. Hojiblanca. The fruits were washed in sterilized water with Tween 20 at 20 μl/liter and surface sterilized in 20% sodium hypochlorite for 2 min. Twenty-five of the immature and 25 mature fruits were wounded with a sterile 0.5-mm-diameter needle, inoculated by immersion in aqueous conidial suspensions (2.2 × 105 conidia ml–1) for 30 min. The same number of immature and mature fruits, wounded and unwounded, were treated only with sterilized Tween 20 water and served as the control. All fruit were incubated at 22 to 24°C and 100% relative humidity. The experiment was repeated twice. Eight days after inoculation, 75% of the mature inoculated fruits showed a general rot with pycnidia developing on the surface of the fruit. After 20 days, the first symptoms of disease were observed on immature olive fruits (2%) and all mature fruits. After 50 days, only 14% of immature inoculated fruits exhibited symptoms of the disease. The fungus caused mummification of mature fruits. D. seriata was reisolated from lesions on all infected fruits. There were no differences in the amount and severity of disease between wounded and unwounded fruits. Control fruits did not display disease symptoms after 50 days. Incidence of olive fruits affected by D. seriata in olive orchards in southern Spain is unknown, although it may be low since it was detected only in 1 of 12 olive orchards sampled for Colletotrichum spp. in 2006. Incidence of fruit rot associated with D. seriata in this orchard was 1.2% of fruits, which also were affected by Colletotrichum. D. seriata has a worldwide distribution and infects numerous fruit trees. In our research, D. seriata is pathogenic on olive fruits but weakly virulent. To our knowledge, this is the first report worldwide of the anamorph of “B.” obtusa causing fruit rot on olives. Reference: (1) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.