Report Of Root Research Articles

First Report of Pythium Root and Stalk Rot of Forage Corn Caused by Pythium arrhenomanes in Japan.

Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan.

First Report of Root and Stem Rot Caused by Phytophthora tentaculata on Mimulus aurantiacus in North America.

Sticky monkey flower plant, Mimulus aurantiacus (Phrymaceae), is a small, perennial shrub that is widely distributed throughout California, especially in coastal and disturbed habitats. It is also found in native plantings in parks and landscapes. In October 2012, nearly all the M. aurantiacus plants grown in a Monterey County, CA nursery for a restoration project were stunted and had dull, yellowish leaves. Roots and stem collars had necrotic, sunken lesions with few feeder roots. Thirty percent of the plants had died. Samples of diseased plants were sent to the CDFA-PPDC Lab and tested positive for Phytophthora sp. using the Agdia ELISA Phytophthora kit (Agdia, Elkhart, IN). A Phytophthora sp. was consistently isolated from the tissue on corn meal agar-PARP (CMA-PARP) (2). Sporangia were spherical to ovoid, papillate to bipapillate and 17 to 42.5 (avg. 27.5) × 12 to 35 (avg. 22.9) μm, with a length/breadth ratio of 1.2:1. Chlamydospores, which were spherical, terminal to intercalary, thin walled and 27.5 to 40 μm, and hyphal swellings formed on CMA-PARP. Spherical oospores, 25 to 36 μm, with primarily paragynous antheridia formed readily on V8 juice agar. rDNA sequences of the internal transcribed spacer (ITS) region of the isolates (GenBank KF667505), amplified using primers ITS1 and ITS4, were 100% identical to Phytophthora tentaculata (CBS 552.96, GenBank AF266775) by a BLAST query (1,3). To assess pathogenicity, exposed root crowns of three 3.78-liter potted M. aurantiacus plants were inoculated with 20 ml of zoospore suspension (2 × 104 ml-1). Plants were maintained in a 23°C growth chamber with a 12-h photoperiod and watered daily. Sterile water was applied to the exposed crowns of three control plants. At 2 weeks, all inoculated plants were wilted with chlorotic foliage. After 3 weeks, the cortical tissue of the crowns and roots was discolored and sloughing and P. tentaculata was recovered on CMA-PARP. P. tentaculata did not grow from the asymptomatic control plants. Inoculations were repeated with similar results. P. tentaculata is a homothallic species in Phytophthora clade 1 that causes crown, root, and stalk rot of nursery plants in Europe and China (1,4). A USDA PERAL analysis lists it as one of the top 5 Phytophthora species of concern to the United States (4). Genera infected with P. tentaculata include Apium, Aucklandia, Chicorium, Chrysanthemum, Delphinium, Gerbera, Lavandula, Santolina, Origanum, and Verbena (4). To our knowledge, this is the first report of P. tentaculata in North America. The source of inoculum of P. tentaculata in California remains unknown. The nursery used seed and cuttings of M. aurantiacus from nearby native areas for propagation, and P. tentaculata was not found in neighboring plant hosts or by baiting soil and water at the nursery. All infected M. aurantiacus material was destroyed. The presence of P. tentaculata in California nurseries could have serious economic impacts on the nursery industry and environmental impacts on susceptible native hosts, if spread into the wildlands.

First report of root and stem rot of soybean caused by Phytophthora sojae in Mato Grosso do Sul State, Brazil

Kaufm. &Gerd. is a widespread problem in the southern region of Brazil,affecting most areas where soybeans are grown. However, itsoccurrence in the central region of the country is unusual. In Brazil,this disease was first observed in Rio Grande do Sul State in 1995(Costamilan et al. Ocorrencia de

First Report of Root and Crown Rot of Wasabi (Wasabia japonica Matsum.) Caused by Phytophthora cryptogea in Michigan.

In September 2011, a Phytophthora sp. was isolated from wasabi (Wasabia japonica Matsum.) grown commercially in hydroponic culture in a large production facility in southwest Michigan. Approximately 20% of the plants were affected, resulting in serious losses for the grower. Plants exhibited severe wilting and root and crown rot, with soft water-soaked lesions on the crown and dark lesions on the roots. Small pieces of root tissue with dark lesions were excised and plated onto potato dextrose agar and unclarified V8 agar plates amended with 25 ppm of benomyl, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of pentachloronitrobenzene. Isolates of a Phytophthora sp. were recovered from root tissue. Isolates produced sporangia abundantly in culture. Sporangia averaged 48 μm long × 34 μm wide and were ellipsoid to ovoid, occasionally obpyriform, and were nonpapillate and noncaducous. Distinct hyphal swellings were noted and chlamydospores were observed rarely in culture. The isolate used for inoculations did not produce oospores alone in culture but was able to produce oospores when paired with an A1 culture of P. capsici and incubated in the dark. Oospores were not observed when the isolate was paired with an A2 culture of P. capsici. No growth was observed at 35°C, and the isolate was identified as Phytophthora cryptogea based on morphological and physiological traits. Pathogen identity was further confirmed using PCR primers specific to P. cryptogea (1). In addition, a BLAST search was conducted using the nucleotide database collection in GenBank comparing our isolate against Phytophthora spp., with 99% sequence similarity to P. cryptogea in two sequenced genes, beta tubulin and cytochrome c oxidase 1 (2). Sequences for the isolate were deposited in the GenBank database under accession numbers JX041520 and JX041521. To fulfill Koch's postulates, six small, potted wasabi seedlings were inoculated by placing 3 g of 1-month-old infested millet (100 g of millet, 72 ml of distilled water, 0.08 g of asparagine, and 10 7-mm diameter V8 agar plugs with actively growing P. cryptogea) onto the soil surface of each pot under coconut coir mulch. Plants were watered heavily after soil infestation and as needed thereafter. Three control plants were inoculated with sterile millet seed. The experiment was repeated once. Wilting was observed within 5 and 7 days, respectively, in the first and second experiment. All six inoculated plants were severely wilted within 25 and 56 days, respectively, except for a single plant in the second experiment that never wilted. Root and crown rot was observed on wilted plants and dark lesions could be observed on root tissue. P. cryptogea was recovered from five of the six plants inoculated in each experiment. None of the control plants in either experiment displayed symptoms of wilting, and the pathogen was not recovered from these plants when pieces of root tissue were excised and plated onto amended V8 agar. To our knowledge, this is the first report of P. cryptogea causing crown and root rot of wasabi.

First Report of Root and Basal Stem Rot Caused by Phytophthora nicotianae on Tree Aeonium (Aeonium arboreum) in Italy

The genus Aeonium, family Crassulaceae, comprises approximately 35 species that are native to northern Africa and the Canary Islands. Tree aeonium (Aeonium arboreum (L.) Webb & Berthel.) is a bushy, perennial succulent with rosettes of tender, waxy leaves at the apex of few-branched or occasionally single, naked stems. Mature rosettes bear yellowish inflorescences. Aeoniums are cultivated as ornamentals in gardens and containers. During the summer of 2009, in a garden in eastern Sicily (southern Italy), 3-year-old potted plants of tree aeonium showed stunting, shrivelling, and chlorosis of leaves and drop of external leaves associated with root and basal stem rot. Drops of an amber exudate oozed from the basal stem. Tissues of the basal stem were soft, but no external necrosis was visible. A species of Phytophthora was consistently isolated from symptomatic roots and basal stem tissues on a medium selective for Oomycetes (2). Axenic cultures were obtained by single-hypha transfers. The pathogen was identified by morphological criteria as Phytophthora nicotianae B. de Haan; it formed stoloniferous colonies on potato dextrose agar and grew between 8 and 38°C, with the optimum at 30°C. On V8 juice agar it produced spherical, intercalary chlamydospores (mean diameter of 26 μm) and persistent, mono- and bipapillate, spherical to ovoid, ellipsoid, obpyriform sporangia that measured 29 to 56 × 22 to 45 μm with a mean length/breadth ratio of 1.3:1. All isolates were A2 mating type and formed spherical oogonia (mean diameter 28 ± 2 μm) with smooth walls and amphigynous antheridia in dual cultures with a reference isolate of the A1 mating type of P. nicotianae. BLAST analysis of the internal transcribed spacer (ITS) region of rDNA of a representative isolate from aeonium (IMI 398812, GenBank Accession No. HQ433333) amplified by PCR using the ITS6/ITS4 universal primers (1), revealed 99% similarity with the sequences of a reference isolate of P. nicotianae available in GenBank (Accession No. EU331089.1). Pathogenicity of isolate IMI 398812 was demonstrated by transplanting cuttings of A. arboreum into pots filled with a mixture of steam-sterilized sandy loam soil and inoculum (4% vol/vol) produced by growing the isolate for 20 days on wheat kernels. Ten plants were transplanted into 3-liter pots (two plants per pot) while 10 plants, transplanted into pots filled with a mixture of steam-sterilized soil and noninoculated kernels, were used as controls. Plants were kept in a greenhouse at 25 to 28°C and watered daily to field capacity. Thirty to forty days after the transplanting into infested soil, cuttings developed the same symptoms observed on plants with natural infections. Control plants remained symptomless. P. nicotianae was reisolated from symptomatic plants, thereby completing Koch's postulates. To our knowledge, this is the first report of P. nicotianae on an Aeonium species worldwide. The economic relevance of this disease is minor because aeoniums are not cultivated on a large scale. Moreover, the disease may be easily prevented by avoiding excess irrigation water since aeoniums need a well-drained soil or potting mix and do not tolerate soil waterlogging.

First Report of Root and Crown Rot of Almond Caused by Phytophthora spp. in Turkey.

Almond (Prunus dulcis) production is currently increasing in Turkey. Losses of approximately 1% associated with root and crown rot of almond seedlings were observed in two commercial nurseries in Ankara and Düzce provinces in 2009. Aboveground symptoms were leaf chlorosis and wilt. Feeder roots were decayed, necrosis occurred on taproots and basal stems, and plants collapsed within several weeks. Roots were washed in tap water and 9 to 10 pieces (3 to 5 mm long) of root tissue taken from the margins of canker lesions, without surface disinfection, were placed on selective medium P5ARPH-CMA (2). Plates were incubated for 3 to 5 days at 20°C in darkness and a number of Phytophthora spp. were recovered. Actively growing mycelium was transferred to carrot piece agar containing β-sitosterol (per liter: carrot piece, 40 g; agar, 20 g; β-sitosterol, 20 mg). Isolates were identified as Phytophthora cactorum and P. citrophthora on the basis of morphological characteristics (1). P. cactorum produced abundant sporangia, oogonia, and paragynous antheridia on carrot piece agar plus β-sitosterol. It had conspicuously papillate and caducous sporangia with short pedicel. Sporangia were usually ovoid but sometimes nearly spherical. P. citrophthora did not produce sexual structures in single culture. It produced papillate, noncaducous sporangia, which were usually ovoid and obpyriform, often asymmetrically shaped and rarely possessed more than one apex. P. citrophthora did not grow at 35°C but it grew well at 30°C. Isolate identities were confirmed by sequence analysis of the ribosomal DNA internal transcribed spacers 1 and 2 (GenBank Accession Nos. HM357622, HM357623, HM357624, HM357625) using primers ITS1 and ITS2 (3). One representative isolate of each species was used to inoculate eight 2-year-old almond plants with an agar plug with actively growing mycelium that was attached to exposed cambium of basal stems. Agar plugs without mycelium were used for eight control plants. All plants inoculated with Phytophthora spp. collapsed within 3 to 4 weeks. Control plants remained healthy. Phytophthora spp. were reisolated from necrotic basal stems. To our knowledge, this is the first report of P. cactorum and P. citrophthora of almond in Turkey.

First Report of Root and Basal Rot of Angelica acutiloba Caused by Fusarium solani in Taiwan.

Angelica (Angelica acutiloba (Siebold. & Zucc.) Kitag.) is one of the most important traditional Chinese medicines in Taiwan. The medicinal herb has been mainly imported from China, but cultivation at a commercial scale has also been established in recent years in Hualien County, Taiwan. In September 2008, angelica plants in a field at Liou-shih-dan Mountain displayed symptoms of yellowing, stunting, rotting of roots and basal stem, and wilting. A severe brown discoloration of vascular tissue along the stems of infected plants was observed. One or more Fusarium spp. was consistently isolated from the roots and stems of diseased plants. Isolates R3, R4, and R5 were incubated for 14 days on celery tissues to produce chlamydospores, and 33 g of celery tissue with chlamydospores were mixed with 500 ml of soil per pot as inoculum. One 4-month-old angelica seedling was planted per pot. Three angelica plants were inoculated with each isolate in the first test and nine plants were inoculated with each isolate in the second test. Other seedlings were inoculated with water as checks. Pathogenicity tests were conducted twice. Incidence of diseased plants was 66, 100, and 33% in the first test, and 66, 100, and 44% in the second test for the R3, R4, and R5 isolates, respectively. Symptoms similar to those on the diseased plants in the field were produced, with leaves turning yellow starting 7 days after inoculation and wilt and discoloration of roots 14 days after inoculation. Fusarium spp. also were reisolated from the diseased plants. Genomic DNA was extracted from mycelium with a fungal genomic DNA purification kit, and the internal transcribed spacer (ITS) rDNA region was amplified and sequenced with primers ITS-4 and ITS-5. The sequence of the resulting ~550-bp amplicon was compared with those in GenBank. The ITS sequences of the R3, R4, and R5 isolates shared 98.7, 98.7, and 97.9% similarity with F. solani isolate AF129104 (3), respectively. Phylogenetic analysis also showed that the three isolates were closer to F. solani than to other Fusarium species. Both macroconidia and microconidia of the R4 isolate were produced on potato dextrose agar. Macroconidia were three to five septate and 27.2 to 37.8 × 4.4 to 6.2 μm; microconidia were zero to one septate and 9.3 to 14.7 × 2.9 to 4.8 μm. Chlamydospores produced on celery juice agar were terminal or intercalary, solitary, in pairs or in chains, and 9.3 to 12.1 μm. Morphological characteristics identified the three isolates as F. solani (Martius) Snyder & Hansen according to Fu and Chang (2) and Chung et al. (1), which agrees with the ITS comparison. To our knowledge, this is the first report of root and basal rot caused by F. solani on angelica in Taiwan.

First Report of Root and Stem Rot Caused by Phytophthora nicotianae on Peperomia tetraphylla in China.

Peperomia tetraphylla, an evergreen herb, is becoming increasingly popular as a potted ornamental plant in southern China. In the summer of 2008, in some commercial flower nurseries in Shenzhen, Guangdong Province, P. tetraphylla showed extensive black stem and root rot, with leaves dropping from the rotten stem. Small pieces (approximately 3 mm2) of stems and leaves were excised from the margins of the black lesions, surface disinfected for 30 s to 1 min in 0.1% HgCl2, plated onto potato dextrose agar (PDA), and incubated at 25°C in the dark. All the plated samples yielded Phytophthora, and microscopic examination of pure cultures grown on PDA plates showed arachnoid colonies with abundant aerial mycelium, chlamydospores, and a few sporangia. Numerous sporangia were formed in sterile soil extract. Sporangia were ovoid or obpyriform, noncaducous, with prominent solitary papillae, and measured 31 to 52 μm (average 38 μm) × 21 to 34 μm (average 27 μm). Chlamydospores were spherical and 21 to 34 μm in diameter (average 28 μm). The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using primers ITS4/ITS5 and sequenced (2). The ITS sequence, when submitted for a BLAST search in the NCBI database, showed 100% homology with the sequences of two reference isolates of Phytophthora nicotianae (Accession Nos. AY833526 and EU433396) and the consensus ITS sequence was deposited in the NCBI as Accession No. GQ499373. The isolate was identified as Phytophthora nicotianae on the basis of morphological and molecular characteristics (1). Pathogenicity of the isolate was confirmed by inoculating 1-year-old plants of P. tetraphylla growing in pots. The isolate was grown for 7 days on PDA plates and mycelial plugs, 5 mm in diameter and taken from the advancing margins of the colonies, were buried approximately 1 cm deep near the base of the stem in such a way that the mycelium on the plugs was in contact with the surface of the stem, which had been wiped earlier with 70% ethanol and gently wounded with a needle. Plants treated the same way but inoculated with sterile PDA plugs served as control plants. Three plants in each pot were inoculated and there were five replications each for the treatment and the control. All plants were kept in a greenhouse at 22 to 32°C. After 6 to 7 days, the inoculated plants showed black lesions around the mycelial plugs; symptoms of root and stem rot developed rapidly thereafter and the plants collapsed within 2 weeks. All symptoms on the inoculated plants were identical to those observed in naturally diseased plants, whereas the control plants remained healthy. The same fungus was consistently reisolated from the inoculated plants. To our knowledge, this is the first report of Phytophthora nicotianae on P. tetraphylla in China.

First Report of Root and Crown Necrosis of Bean Caused by Pythium aphanidermatum in Spain.

Adult bean plants (Phaseolus vulgaris cv. Festival) growing in a commercial greenhouse in southeastern Spain developed symptoms of root necrosis, necrotic streaks on the basal stems, and plant wilt. A Pythium sp. was isolated consistently from roots and basal stems on selective agar (P5ARP). Single-hyphal transfers produced intercalary antheridia, oogonia (23 to 26 μm in diameter), oospores (18 to 20 μm in diameter), and zoospores in toruloid sporangia. Cardinal temperatures were a minimum of 10°C, an optimum of 28 to 34°C, and a maximum of 40°C. Daily growth rate on corn meal agar at 25°C was 15 mm. The internal transcribed spacer 1 (ITS 1) rDNA sequence of the isolate matched the sequences of Pythium aphanidermatum in GenBank. The sequence of isolate Py-294 was deposited in GenBank, Accession No. AM396563. This isolate was identified as P. aphanidermatum on the basis of morphological and cultural characteristics (1) and the ITS rDNA sequence. To fulfill Koch's postulates, 50 ml of inoculum of isolate Py-294 was used to inoculate bean plants (cvs. Donna and Emerite) at the five-leaf stage. The inoculum was prepared by homogenizing 2-week-old potato dextrose agar-petri plate cultures in 300 ml of distilled water. The plants were maintained in a greenhouse at temperatures of 18.8 to 30.3°C. Irrigation water had an electric conductivity of 0.5 to 0.6 dS·m-1 while the nutrient solution had 1.9 to 2.1 dS·m-1. Two months after sowing, 35.4 and 100% of cvs. Donna and Emerite, respectively, developed root necrosis, while control plants grown in bags containing noninoculated perlite remained healthy. The pathogen was reisolated from roots and basal stems of symptomatic plants. The test was repeated with similar results. To our knowledge, this is the first report of P. aphanidermatum as the causal agent of root and crown necrosis of adult bean plants in Spain. Reference: (1) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:242, 1981.

First Report of Root and Collar Rot by Phytophthora cryptogea on Sweet Cherry in Italy.

Sweet cherry (Prunus avium L.) cultivation has increased in Italy and ≈30,000 ha are now in production. In the spring of 2006, a disease survey was conducted in orchards of central Italy. Decline symptoms of Prunus avium 'Durone' grafted on wild rootstocks of Prunus avium consisted of reduced tree vigor, yellowing of leaves, and development of root and collar rot. A reddish brown discoloration of the inner bark and wood was observed on symptomatic trees. Symptoms were recorded in three cherry orchards seasonally submitted to soil saturation conditions. In one orchard, symptoms were observed on approximately 30% of 170 surveyed trees. A Phytophthora species was consistently isolated on PARPH-V8 medium (2) from collar lesions of five cherry trees over nine trees randomly chosen among symptomatic trees in the three orchards. Colonies (20) were isolated and all appeared fluffy on potato dextrose agar and did not grow at 35°C. The morphological characteristics fit the descriptions of Phytophthora cryptogea Pethybr. & Lafferty (1). Sequence analysis of the internal transcribed spacers region of isolates shared 100% homology with P. cryptogea (GenBank Accession Nos. EF418948.1, EF418943.1, and EF153671.1). Pathogenicity of two P. cryptogea isolates (AN1 and AN2, Accession Nos. EF661576 and EF661577, respectively) was tested by soil infestation in a growth chamber at 22°C using 15 1-year-old rooted seedlings of wild rootstock of Prunus avium for each isolate. Noninoculated seedlings (15) were used as negative controls. The inoculum was produced by growing isolates on autoclaved millet grains moistened with V8 juice for 4 weeks. Each plant was inoculated with 30 cm3 of inoculum per liter of soil. Each seedling was randomly assigned to isolates AN1, AN2, or the noninoculated control and arranged in a complete random design. All plants were flooded for 24 h every 2 weeks for 8 weeks. At the end of the experiment, all inoculated seedlings showed crown wilting and root rot. Control plants showed no symptoms. Percentage of necrotic lateral roots expressed as dry weight (±SE) was 19.9 ± 0.6, 17.5 ± 0.2, and 4.4 ± 0.5, respectively, for seedlings inoculated with isolates AN1, AN2, and controls (analysis of variance F = 7.48, P < 0.01). Values of inoculated seedlings were significantly different from the control plants with the Tukey Post-Hoc test. No difference in virulence was observed between the two isolates. P. cryptogea was always reisolated from the roots of inoculated plants. Although pathogenicity of P. cryptogea on sweet cherry has been reported in the United States and other European countries (3,4), to our knowledge, this is the first record of P. cryptogea on sweet cherry orchards in Italy. The presence of P. cryptogea in cherry orchards is of significant concern because of its aggressiveness to this host and woody hosts such as apple, apricot, peach, walnut, and kiwi.

First report of Bipolaris sacchari causing wheat stem‐base disease in Iran

During May and June 2003, wheat plants in several fields in Hamada, Iran showed symptoms of root rot and crown necrosis. Stem-base and root samples were submerged in 0·5% sodium hypochlorite for 3–5 min, rinsed with sterile distilled water, transferred to Petri dishes containing potato dextrose agar (PDA), and incubated at 22–25°C for 7 days. Bipolaris spp. was the most prevalent fungus isolated. Based on the host origin, conidia and conidiophore morphology, number of pseudosepta and hilum characteristics, the pathogen was identified as Bipolaris sacchari (Sivanesan, 1987). Inoculum for pathogenicity tests was produced by seeding 200 g sterile wheat stubble in an Erlenmeyer flask with four 5-mm-diameter mycelial plugs from 5-day-old fungal colonies grown on PDA, and incubated at room temperature (20°C) for 3 weeks. The inoculated wheat straw was incorporated into sterile soil (1% w/w), then transferred to five 25-cm pots and planted with eight wheat seeds per pot. Control plants were grown in soil containing 1% sterile wheat straw. The treatments were replicated five times. Plants were grown in a glasshouse for 75 days at 24°C, then scored for disease (Singleton et al., 1992). Symptoms were similar to those observed in the field, and no disease was found in the control plants. The fungus was reisolated from the infected parts. In a separate study using wheat cvs Alvand and Roushan, the pathogen caused a significant reduction in seed germination and subsequent plant height. This is the first report of root and stem rot disease of wheat caused by B. sacchari in Iran. Further field studies are needed on the ecology and pathogenicity of B. sacchari to formulate steps for controlling root and stem rot of wheat. Financial support for this work by the Research Council of Bu-Ali Sina University, Hamadan, Iran, is gratefully acknowledged.

Root and Foot Rot on Tomato Caused by Phytophthora infestans Detected in Belgium.

In January 2003, a severe root and foot rot was observed on 2-month-old wilted tomato (Lycopersicon esculentum Mill.) plants in a large-scale (2.5 ha) commercial greenhouse setting in Belgium. Tomato plants (10%) produced from healthy nursery-grown seedlings and planted to new, clean rockwool and drip irrigation with UV-disinfected water developed symptoms. Symptom development was restricted to lower plant parts with severe rotting of the entire root system and dark lesions girdling the stem base. No symptoms of disease were observed on the foliage or upper stems. Cross sections of the stem base revealed brown discoloration of internal tissue, including the vascular tissue and pith. Dark brown lesions also occurred on the roots. Sections of the stem base, the upper roots (sampled near to the stem base), and the lower roots (sampled on roots deeper in the rockwool) were plated separately on corn meal agar. The oomycete pathogen Phytophthora infestans (Mont.) de Bary was identified in each sample on the basis of morphological characteristics observed directly with light microscopy. Branched sporangiophores with slight swellings and characteristic lemon-shaped sporangia (35 × 20 μm and ratio length/width of 1.75 μm) at their tips were obvious after incubation in darkness at 24°C. Oospores and chlamydospores were not observed. After multiple soil treatment with oomycete-specific fungicides, the plants recovered. Since the occurrence of P. infestans on roots is unusual, the identity of the pathogen on the diseased plant tissues was confirmed with three techniques, DNA array identification, internal transcribed spacer (ITS) sequencing, and a polymerase chain reaction (PCR) amplification using P. infestans-specific primers. DNA was directly processed from separate samples of upper and lower root and stem base tissue. The DNA array used was originally developed to detect and identify the key fungal pathogens of tomato (2). Among detector probes for other tomato pathogens, this array contains oligonucleotide detector probes for P. infestans (PIN1: 5'-GGT TGT GGA CGC TGC TAT T and PIN2: 5'-AAT GGA GAA ATG CTC GAT TC). These probes are based on ITS sequences (ITS I and ITS II). Using conserved ribosomal primers OOMUP18Sc (5'-TGC GGA AGG ATC ATT ACC ACA C) and ITS4, oomycete DNA was amplified by PCR and simultaneously labeled with alkaline-labile digoxigenin (2). All generated amplicons strongly hybridized to the oligonucleotide detector probes for P. infestans and not to any other pathogen-specific detector probe present on the array. The pathogen could not be detected in roots and stem bases of symptomless plants. In addition, the ITS-region was sequenced and showed 100% homology with multiple GenBank accessions of P. infestans sequences. As a third confirmatory test, a PCR was performed on DNA extracts from infected root and stem base tissues using a primer set specific to P. infestans (O8-3/O8-4 [1]). A band of the expected size was produced for the infected stem base and root samples. Until now, this pathogen was known worldwide to cause late blight on potatoes and tomatoes. To our knowledge, this is the first report of root and foot rot of tomato caused by P. infestans.

First Report of Root and Crown Rot Caused by Phytophthora cinnamomi Affecting Native Stands of Arctostaphylos myrtifolia and A. viscida in California.

Ione manzanita (Arctostaphylos myrtifolia) is a rare, endemic, evergreen shrub restricted to Ione formation soils (infertile, acidic, sedimentary oxisols) in the foothills of the Sierra Nevada. The widely distributed A. viscida (whiteleaf manzanita) intermixes with A. myrtifolia at the margins of Ione formation soils. In 2002, we observed extensive mortality within two mixed stands of A. myrtifolia and A. viscida near Ione, CA. At one site, nearly all plants of both species in a 0.25-ha area had died recently. At a second site, most of the A. myrtifolia and A. viscida plants on several hectares died at least 5 years earlier. Dying plants of both species exhibited wilting and desiccation of the foliage; dark brown discoloration and necrosis of the root crown, taproot, and some large roots; and loss of fine roots. Plants of all age classes were affected. We consistently isolated a Phytophthora sp. from symptomatic plants of both species using PARP (1) and acidified potato dextrose agar. We recovered the same Phytophthora sp. from soil collected under dead plants using green pears to bait flooded soil samples. The pathogen was not recovered from soil collected under healthy plants 50 m from the nearest dead plant. Based on the morphology of the hyphae, chlamydospores, sporangia, and the sequence of the internal transcribed spacer rDNA, we identified the pathogen as P. cinnamomi Rands (GenBank Accession No. AY267370; ATCC No. MYA-2989). To test pathogenicity, we poured zoospore suspensions (4 × 104 zoospores per pot) on the soil of eight pots with rooted A. myrtifolia cuttings and four pots with rooted A. viscida cuttings (1 14-month-old plant per pot). The soil in inoculated and uninoculated control pots (eight A. myrtifolia and two A. viscida) was flooded for 20 to 23 h. All inoculated A. myrtifolia developed severe root and crown rot, and seven of eight died within 17 days. All inoculated A. viscida developed severe root rot, and three of four developed 5- to 10-cm long basal cankers. After 17 days, we isolated P. cinnamomi from inoculated A. myrtifolia (eight of eight) and A. viscida (two of four) but not from controls, which remained healthy. We tested pathogenicity in native soil by transplanting rooted cuttings (eight A myrtifolia and six A. viscida) into pots of naturally infested soil from one of the disease centers. Controls (four and three plants, respectively) were planted in soil collected from under healthy plants. Pots were flooded for 12 to 13 h for 11 days (A. myrtifolia) or 6 weeks (A. viscida) after transplanting. All plants grown in naturally infested soil developed root and crown rot, and all A. myrtifolia and one A. viscida died within 5 weeks of transplanting. Plants grown in field soil collected near healthy plants remained asymptomatic. We isolated P. cinnamomi from all eight A. myrtifolia and three A. viscida plants grown in infested soil but not from the controls. To our knowledge, this is the first report of root and crown rot caused by P. cinnamomi on A. myrtifolia and A. viscida. P cinnamomi was first isolated in the state in 1942 (2), but it has not previously been reported to caused significant mortality in natural stands of California native species. This disease will significantly impact conservation of the already threatened A. myrtifolia.

First Report of Root and Crown Rot of Sage Caused by Phytophthora cryptogea in Italy

Sages are cultivated as aromatic and ornamental plants in Italy and represent the common name of certain species of Salvia and Phlomis (family Lamiaceae). In Sicily (southern Italy) during the summer of 2001, ≈40% of 1,400 2-year-old landscape plants of S. leucantha Cav. (Mexican bush sage or velvet sage) showed symptoms of stunting, chlorosis, and gradual dieback or sudden wilt, which are associated with root and crown rot. Plants were supplied by a commercial nursery, transplanted from pots in the spring, and irrigated using a trickle system. Phytophthora was isolated consistently from roots and basal stems of symptomatic plants on a BNPRAH medium (2). The species was identified as P. cryptogea Pethybr. & Laff., primarily on the basis of morphological and cultural characteristics. Five representative single-hypha isolates were characterized. On potato dextrose agar, they formed colonies with a slight petaloid pattern. Cardinal temperatures for mycelium growth were 2°C, minimum; 25°C, optimum; and 30 to 35°C, maximum. Hyphal swellings were abundant in aqueous culture. Sporangia were obpyriform, persistent, nonpapillate, and proliferous (2). All isolates were the A1 mating type and formed oogonia, amphigynous antheridia, and oospores in dual cultures with reference isolates of the A2 mating type of P. cryptogea and P. drechsleri. Identification was confirmed by electrophoresis of mycelium proteins on a polyacrylamide slab gel (1). Electrophoretic patterns of total soluble proteins from the sage isolates were identical or very similar to those from 10 reference isolates of P. cryptogea from various hosts, including isolate IMI 180615 (ex-type isolate). Conversely, the electrophoretic pattern of the isolates of P. cryptogea from sage was clearly distinct from those from reference isolates of other species included in Waterhouse's taxonomic group VI. Esterase (EC 3.1.1.2.) zymograms of the sage isolates corresponded to those of isolates of P. cryptogea included in electrophoretic group 2 (1). The pathogenicity of a representative isolate of P. cryptogea from sage was tested in the greenhouse using 4-month-old plants of Mexican bush sage. Inoculum was produced on a mixture of vermiculite and autoclaved oat seeds (4) and mixed with steam-sterilized sandy loam soil at a concentration of 4% (vol/vol). Plants were transplanted in pots (12 cm diameter) filled with infested soil; control plants were grown in pots containing noninfested soil. After transplanting, all pots were placed in shallow trays filled with water for 24 h to saturate the soil. All plants grown in infested soil showed extensive root necrosis and dieback ≈30 days after transplanting, and P. cryptogea was reisolated from roots of symptomatic plants. Control plants did not develop symptoms. Root and crown rot of sage caused by P. cryptogea has been reported previously in California (3). To our knowledge, this is the first report of P. cryptogea on sage in Italy. Root rot caused by P. cryptogea may be a potential problem for commercial cultivation of sage as no serious disease of this plant has been reported in Italy so far.

First Report of Root and Collar Rot by Sclerotium rolfsii on Apple Trees in Italy.

Sclerotium rolfsii Sacc. (teleomorph Athelia rolfsii (Curzi) Tu & Kimbrough) is a polyphagous, soilborne plant pathogen. In summer 1998, a sudden death of 2-year-old apple trees (Malus domestica Borkh.) cv. Royal Gala grafted on M9 rootstock was observed in an orchard near Rome, Italy. Symptoms were stunted vegetation, leaf chlorosis, and root and collar rot. A fungus identified as S. rolfsii was observed producing sclerotia and whitish mycelial strands on root and collar bark. Isolations from roots and at the margin of subcortical necrosis on the collar consistently yielded S. rolfsii colonies on potato dextrose agar (PDA); sclerotia developed within 7 days. Koch's postulates were fulfilled by inoculating 10 1-year-old apple tree cv. M9 rootstocks, grown in 3.5-liter pots, with an S. rolfsii isolate grown for 1 week on PDA at 25°C. One ground plate per plant was used, placed around collar and main roots. Five control plants were treated with PDA only. Rootstocks were kept in the greenhouse at 26 ± 2°C. Within 2 months, 70% of inoculated plants died, with marked necrosis girdling the collar. The other inoculated plants showed a general decline, with widespread necrosis on collars and main roots. Control plants remained healthy. S. rolfsii was reisolated from collars and roots of symptomatic plants. S. rolfsii has been recorded on apple trees in the U.S., India, China, and Israel. In Italy, it is destructive on several crops, and was recently recorded on walnut (1). This first outbreak of S. rolfsii on apple in Italy may have been favored by exceptionally warm late spring and summer temperatures. Reference: (1) A. Belisario and L. Corazza. Plant Dis. 80:824, 1996.

INCIDENCE AND CLINICAL SIGNIFICANCE OF CORONARY ARTERY DISEASE IN DIABETES MELLITUS

THE INCIDENCE of coronary artery disease in diabetic persons has been investigated repeatedly at autopsy, with general agreement that severe coronary arteriosclerosis is more frequent in the diabetic than in the nondiabetic patient.1Of 316 patients with diabetes over the age of 40 included in the report of Root, Bland, Gordon and White,1a38.2 per cent of the men and 32.2 per cent of the women had coronary artery occlusions. The same study demonstrated such occlusions in only 9.9 per cent of 1,521 nondiabetic men and in 4.9 per cent of 789 nondiabetic women, all over the age of 40. In the same age group Enklewitz1bfound coronary artery occlusions in 31.8 per cent of 261 persons with diabetes. In the hearts of a group of unselected patients studied by an improved technic of injection plus dissection for the detection of pathologic alterations in the coronary arteries,