First Report of Citrus Root Rot Caused by Phytophthora palmivora in Egypt

During spring-summer 2009, a survey was conducted to determine the species of Phytophthora present in citrus nurseries in Egypt. A total of 300 samples of soil and fibrous roots were collected from the rhizosphere of symptomatic Volkameriana lemon (Citrus volkameriana Tan. & Pasq.) plants growing in Delta (Benha-Qalyubia) and a desert (Cairo/Alexandria desert road) citrus nurseries. Plants showed various symptoms. Canopies of affected plants showed few and yellowish leaves, a general stunted growth, no new vegetation, and sometimes sudden desiccation; the root system showed few dark brown feeder roots, no new yellow-white apexes, and a fibrous appearance of the rootlets due to disintegration of the cortical bark but not of the xylem. Collected rootlets and soil were plated in Petri dishes containing a selective medium for the oomycete Phytophthora (2) and incubated for 3 to 6 days at 19 ± 1°C as described by Ippolito et al. (1). Pure cultures were obtained by single-hypha transfers and the isolates were identified as Phytophthora palmivora (Butler) Butler on the basis of morphological and cultural characteristics (3). Isolates formed stoloniferous colonies on potato dextrose agar (PDA) and grew between 10 and 30°C, with the optimum at 25°C. On V8 juice agar, they showed a highly fluffy pattern and produced terminal and intercalary globose chlamydospores. Sporangia were papillate, elliptical (45 to 51 × 29 to 35 μm; length/breadth ratio of 1.3:1.8), and were caducous with short pedicel. All isolates were A2 mating type, forming spherical oogonia and amphigynous antheridia in dual cultures with reference P. palmivora isolate of A1 mating type. Identification of the isolates was further confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region using the universal primers ITS4 and ITS6. BLASTn analysis of ITS sequences (GenBank Accession No. HE583183) showed 99% homology with P. palmivora isolates available in GenBank. Pathogenicity tests for P. palmivora were conducted by inoculating three groups of ten 6-month-old Volkameriana lemon plants, transplanted into 1.4 liter pots with growing medium artificially inoculated at the rate of 1% (v/v) of P. palmivora inoculum produced according to Yaseen (4). Ten uninoculated plants served as a control. Two months after the inoculation, plants were analyzed for canopy symptoms and the presence of pathogen in feeder roots. More than 50% of inoculated plants showed foliage symptoms and extensive decay of feeder roots. Colonies of Phytophthora were recovered from necrotic rootlets and identified as P. palmivora, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of P. palmivora as a pathogen to citrus plants in the Egyptian nurseries. P. palmivora should be considered a potential threat to the Egyptian citrus industry since it may negatively influence the nurseries and orchards production in the future. References: (1) A. Ippolito, V. De Cicco, and M. Salerno. Rivista di Patologia Vegetale 2:57, 1992. (2) H. Masago, M. Yoshikawa, M. Fukada, and N. Nakanishi. Phytopathology 67:425, 1977. (3) D. J. Stamps. Revised tabular key to the species of Phytophthora. CAB International Mycological Institute, Kew, Surrey, 1990. (4) T. Yaseen. Molecular diagnosis and biological control of Phytophthora-citrus root rot. PhD thesis. University of Bari, Italy, 2004.

First Report of Cucurbit aphid-borne yellows virus in Iran Causing Yellows on Four Cucurbit Crops

A survey was conducted from 2001 to 2004 in the major cucurbit-growing areas in Iran to reassess the relative incidence of cucurbit viruses. Severe yellowing symptoms were observed frequently on older leaves of cucurbit plants in various regions in outdoor crops, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) (1,2). Leaf samples (n = 1019) were collected from plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita sp.), and watermelon (Citrullus lanatus L.) showing various virus-like symptoms (mosaic, leaf deformation, yellowing). All samples, collected from 15 provinces, were screened for the presence of CABYV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with IgGs and alkaline phosphatase-conjugated IgGs against a CABYV reference isolate (1). Of the 1,019 samples tested, 471 were positive for CABYV using DAS-ELISA. Some of the positive samples had typical severe yellowing symptoms while symptoms in other samples were masked by mosaic or leaf deformations caused by other viruses frequently found in mixed infections (data not shown). During the entire survey, CABYV was detected by DAS-ELISA in 201 of 503 melon samples, 72 of 129 cucumber samples, 158 of 249 squash samples, and 40 of 138 watermelon samples. These results indicate that CABYV is widely distributed on four cucurbit species in the major growing areas of Iran. In order to confirm CABYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St Louis, MO) were obtained from 25 samples that were positive using DAS-ELISA originating from Khorasan (n = 4), Esfahan (n = 6), Teheran (n = 3), Hormozgan (n = 4), Azerbaiejan-E-Sharqi (n = 4), and Kerman (n = 4). Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using forward (5′-CGCGTGGTTGTGG-TCAACCC-3′) and reverse (5′-CCYGCAACCGAGGAAGATCC-3′) primers designed in conserved regions of the coat protein gene according to the sequence of a CABYV reference isolate (3) and three other unpublished CABYV sequences. RT-PCR experiments yielded an expected 479-bp product similar to the fragment amplified with extracts from the reference isolate. No amplification of the product occurred from healthy plant extracts. To our knowledge, this is the first report of the occurrence of CABYV in Iran on various cucurbit species. The high frequency (46.2%) with which CABYV was detected in the samples assayed indicates that this virus is one of the most common virus infecting cucurbits in Iran. References: (1) H. Lecoq et al. Plant Pathol. 41:749, 1992 (2) M. A. Mayo and C. J. D'Arcy. Page 15 in: The Luteoviridae. H. G. Smith and H. Barker, eds. CAB International Mycological Institute, Wallingford, UK, 1999. (3) H. Guilley et al. Virology 202:1012, 1994.

Dasylirion serratifolium as a New Host of Botrytis cinerea, the Causal Agent of Leaf Spots and Blight in Italy

Sandpaper sotol (Dasylirion serratifolium Zucc.), native to Mexico, has green leaves with margins highlighted by whitish yellow prickles like a fine sandpaper. During the spring of 2004 and 2005, necrotic lesions were observed in the middle of leaf blades and near prickles on 2- to 5-year-old, container-grown sandpaper sotol plants from two nurseries in eastern Sicily (Italy). Symptoms were detected on 20 to 30% of plants and consisted of reddish lesions that developed a reddish brown stripe surrounded by a yellow halo. As lesions enlarged, the center turned yellow and expanded rapidly causing blight of young leaves. Occasionally, symptomatic tissues had masses of gray fungal conidia and/or sclerotia. Botrytis cinerea was isolated consistently from infected tissues disinfected for 1 min in 1% NaOCl and rinsed in sterile distilled water and grown on potato dextrose agar (PDA). Hyaline, ovoid conidia (average 6.4 × 9.7 μm) and conidiophores were similar to those described of B. cinerea, and 5- to 8-day-old cultures developed black sclerotia that were round or irregular in shape (average 1.55 × 1.02 mm) that is typical of gray mold (1). Koch's postulates were performed by spraying 6-week-old sandpaper sotol plants grown in 12-cm pots with a spore suspension (1 × 106 CFU per ml) obtained from 12-day-old cultures grown on PDA. Eight plants were naturally wounded by scratching leaf blades among themselves and were subsequently inoculated, while eight plants were inoculated without wounding. An equal number of noninoculated plants sprayed with sterile water served as controls. All plants were maintained in high humidity conditions (90 to 95% relative humidity) at 20 ± 2°C. Leaf spots similar to the ones observed in nurseries were evident on all naturally wounded and nonwounded plants within 2 to 3 weeks after inoculation. Noninoculated control plants were symptomless. B. cinerea was reisolated from affected tissues. The pathogen has reduced commercial value of sandpaper sotol plants and may represent a limiting factor for the cultivation of this plant in eastern Sicily. To our knowledge, this is the first report in the world of leaf spot and blight caused by B. cinerea on D. serratifolium. Reference: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CAB International Mycological Institute, Kew, Surrey, England, 1971.

First Report of Phytophthora cinnamomi on Ilex glabra in Virginia

During 2002, two nurseries in southeastern Virginia reported losses exceeding 75% of container-grown inkberry holly (Ilex glabra) cv. Shamrock. The development of necrotic leaf spots and blotches followed initial symptoms of leaf yellowing and wilting. Affected leaves rapidly turned brown and fell. Dark brown-to-black roots were washed and plated on agar media. Phytophthora cinnamomi Rands was consistently isolated and identified on the basis of its morphology (2) and single-stranded conformational polymorphism fingerprint (1). The organism had nonpapillate, internally proliferating, noncaducous, ovoid to ellipsoid sporangia that formed only in water. It did not grow at 35°C and had abundant botryose hyphal swellings, coralloid hyphae, and grape-like clusters of chlamydospores. The isolate, determined to be the A2 mating type, produced elongate cylindrical, amphigynous antheridia and oogonia with a tapered base. A pine bark potting mix amended with V8 juicetreated vermiculite colonized by the suspected pathogen was placed in 12-liter containers. Two inkberry holly cv. Shamrock liners were planted in each of three containers and two 1-yr-old plants were planted in each of three additional containers during April 2004. An identical set of six containers of noninoculated plants was also established. During June 2004, inoculated plants exhibited symptoms identical to those observed in nurseries, and P. cinnamomi was isolated. Noninoculated check plants did not develop symptoms. Japanese holly (I. crenata) was previously known as a host, but to our knowledge, this is the first report of inkberry holly (I. glabra) susceptibility. References: (1) P. Kong et al. Fungal Genet. Biol. 39:238, 2003. (2) D. J. Stamps et al. Mycol. Pap. No. 162. CAB International Mycological Institute, Wallingford, UK, 1990.

First Report of Powdery Mildew Caused by Podosphaera leucotricha on Prunus africana in Kenya

Prunus africana, formerly known as Pygeum africanum, is widely distributed in moist, tropical Africa and produces durable timber. Extracts from its bark are used in treatment of prostate disorders. Powdery mildew was observed on nursery-grown seedlings of P. africana in Kenya (Nyeri, Kiambu, and Kericho districts) in the dry seasons of 1998, 1999, and 2000. White ectotrophic mycelial growth was observed on leaves. The fungus caused stunting, distortion of leaves, surface necrosis of invaded tissues, and general decline in growth of seedlings that led to premature leaf fall and death. Invaded leaflets wilted and dropped, leaving behind a bare stem. The primary mycelium was hyaline, with no secondary brown mycelium. The conidial state was conspicuous, with conidia produced in chains. Appressoria were unlobed and nipple shaped. Conidiophores were straight and three-celled, measuring 75 to 112 μm. Conidiophore foot cells were topped by a longer cell and one or two shorter cells measuring 35 to 77 μm. Conidia had fibrosin bodies, were ovoid, and measured 27 to 30 × 17 to 18 μm. The fungus was identified by the International Mycological Institute IMI (W6496) as Podosphaera leucotricha (Ellis & Everh.) E. S. Salmon. Infected leaves of P. africana were deposited at the East African Herbarium, National Museums of Kenya (Accession No. KM-KEFRI/446/2001). Pathogenicity was confirmed by inoculating seedlings of P. africana by gently pressing infected leaves with abundant sporulation onto healthy leaves. The plants were then incubated under moist conditions for 48 h and thereafter maintained in a glasshouse. After 15 days, powdery mildew symptoms developed on seedlings. Examination of leaves confirmed that they were infected with Podosphaera leucotricha. Uninoculated control plants were free of powdery mildew. To our knowlege, this is the first report of Podosphaera leucotricha as a pathogen of P. africana. Reference: 1. H. J. Boesewinkel. Bot. Rev. 46:167, 1980.

Occurrence and Virulence of Bipolaris hawaiiensis on Bermudagrass (Cynodon dactylon) on Poultry Waste Application Sites in Mississippi

Bipolaris hawaiiensis has been reported on bermudagrass (Cynodon dactylon) and other Cynodon spp. from subtropical areas around the world (2). This pathogen has not previously been reported on bermudagrass in North America (1) nor has its virulence been compared with that of other Bipolaris spp. on this host. In July and October 1999, frequencies of dematiaceous hyphomycetous pathogens in live but symptomatic leaves of bermudagrass were determined on two poultry waste application sites in Smith and Covington counties, MS, where foliar disease symptoms were widespread. Common bermudagrass was being grazed in Covington County, and cv. Alicia was being grown for hay in Smith County. At each date and site, 100 stems with leaves exhibiting symptoms of chlorosis and necrosis were collected, and a single leaf with well-developed symptoms from each stem was assayed for pathogens by surface-disinfesting, plating on water agar, and observing fungal sporulation. Multiple species of pathogens were detected on most leaves. Identities and mean frequencies of observed pathogen species across both sites and sampling dates were Exserohilum rostratum (62%), Bipolaris cynodontis (98%), Curvularia lunata (28%), C. geniculata (20%), B. spicifera (3%), and B. hawaiiensis (3%). B. hawaiiensis was detected at both sites and on both sampling dates. It was distinguished from B. cynodontis by smaller conidia (14 to 28 μm long) and from B. spicifera by more than three pseudosepta per conidium. Virulence of B. hawaiiensis on bermudagrass, compared with B. cynodontis and B. spicifera, was assessed in two identical inoculation experiments using three pathogen-inoculated treatments plus an uninoculated control. In each experiment, foliage of 12-week-old plants in five replicate pots per treatment was sprayed with 4 × 104 conidia per ml of water of each pathogen. The pots were incubated under 12-h plant-growth lights at 25°C for 3 days in a water-saturated atmosphere to initiate infection and then grown for seven additional days in ambient air under plant-growth lights at 25°C. All three pathogens induced symptoms of chlorosis and necrotic lesions. Symptoms induced by B. hawaiiensis were similar in severity to those produced by B. spicifera and less severe than those produced by B. cynodontis. To our knowledge, this is the first report of B. hawaiiensis on bermudagrass in North America. The site in Smith County also apparently represents its northernmost known point of occurrence on this continent (2). References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (2) A. Sivanesan. Graminicolous Species of Bipolaris, Curvularia, Drechslera, Exserohilum and their Teleomorphs. Mycol. Pap. No. 158, CAB International Mycological Institute, Wallingford, U.K., 1987.

First Report of Colletotrichum acutatum in Strawberry in Norway

Anthracnose caused by Colletotrichum acutatum J. H. Simmonds was detected in strawberry (Fragaria × ananassa Duch.) for the first time in Norway in 1999. Symptoms were found in greenhouse grown strawberries in the cultivar Korona. Symptoms were typical of strawberry anthracnose: sunken, brown, and firm lesions appeared on maturing fruits. Masses of conidia were produced in acervuli in the center of lesions. The fungus was isolated on acidified potato dextrose agar. Colonies grown on potato dextrose agar (PDA) were pale to mouse gray and became dark greenish to blackish in reverse. Conidia were formed in orange to salmon pink masses in the center of the culture. Conidia in cultures were 16.5 (13.8 to 18.8) × 4.5 (3.8 to 5) μm, and were hyaline, cylindrical, with pointed ends, and aseptate. Setae were never observed in culture or on fruits. The fungus did not form an ascigerous stage in culture. Mycelial growth rate at 25 to 26°C on PDA was 8.1 to 8.4 mm per day. Morphological characters and growth rate were in accordance with previous reports on C. acutatum (1,2). The isolated fungus was confirmed to be C. acutatum by both the International Mycological Institute, Egham, England, and Centraalbureau voor Schimmelcultures, Baarn, the Netherlands. Koch's postulates were fulfilled by inoculating ripe and unripe fruits on strawberry plants with the isolated fungus. Fruits were either sprayed with a conidial suspension (106 conidia per ml) or slightly wounded with a needle that had been dipped in a conidial mass from a pure culture of C. acutatum. Symptoms appeared after 4 days at 20°C, and after 5 days, brown, sunken, circular lesions reached a size of 1 cm in diameter on wounded, ripe fruits. In unripe fruits the lesions developed more slowly, and in unwounded fruits sprayed with a conidial suspension, large, irregular spots developed. Leaves were inoculated by placing a small block of agar at the base of petioles on intact strawberry plants. The tissue underneath the agar was either unwounded or slightly wounded with a needle. After 20 days (at 20 to 25°C) some necrosis developed on both unwounded and wounded petioles. No symptoms were observed in the crown tissue where the inoculated petioles were attached. The fungus was readily reisolated from both fruits and petioles, after which typical morphological characters developed in culture as described above. References: (1) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (2) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990.

New Record of Ceratocystis fimbriata Causing Wilt of Pomegranate in India

Wilt of pomegranate (Punica granatum L.) was first noticed in two areas of the Bijapur district (16°49′N; 75°43′E) of India in 1990. Around 1993, rapid spread of this disease was observed in the entire Bijapur district. The cause was not identified until 1995. Initial symptoms were yellowing and wilting of leaves on one to several branches leading to death of affected plants in a few weeks. Cross sections of diseased plants revealed brown discoloration in the outer xylem from roots to the main trunk. A survey of 44 locations from 1995 to 1998 showed an approximate loss of Rs. 30 lakhs (ca. US$69,770) and 7.5% (3,474 of 47,096 plants wilted) of the crop. At 13 locations, plants also were severely infested with shot hole borer (Xyleborus spp.). In 1996, the fungus, a Ceratocystis sp., was isolated from discolored stem, root, and branch tissues on wilted plants collected from various locations, e.g., A. Sangapur, Bagalkot, Bijapur, Bilagi, Kanamadi, Tajpur, and Tikota. The fungus isolated from Bagalkot was confirmed by the International Mycological Institute (UK) as C. fimbriata Ellis & Halst. (Specimen No. W 5496, PBUR) in 1997; the strain of this fungus, i.e., Latin American group, was identified in 1998 by T. C. Harrington (Iowa State University). Morphological characteristics of mycelium, conidia, conidiophore, chlamydospores, perithecia, and ascospores were similar to those described previously (1). Pathogenicity of this fungus was confirmed by inoculating wounded roots. This is the first report of C. fimbriata causing wilt on pomegranate. Reference: (1) Anonymous. C.M.I. Descriptions of Pathogenic Fungi and Bacteria. No. 141. CAB, Surrey, England.