First Report of Leaf and Stem Downy Blight of Longan Seedlings Caused by Peronophythora litchii in Taiwan.

Fruit downy blight caused by Peronophythora litchii Chen ex Ko et al. is an important disease of litchi (Litchi chinensis Sonn.) in Taiwan, especially in rainy seasons. Previous records indicate litchi as the only natural host of P. litchii, but this pathogen was found on seedlings of longan (Euphoria longana Lamarck) in 2000. Young seedlings of longan that had emerged in a litchi orchard near Caotun, Nantou County, showed symptoms of droopy leaves and leaf blight. Water-soaked lesions appeared on young leaves, which turned into brown, round or irregular lesions (about 3 to 5 cm long). Diseased leaves withered and collapsed eventually. Dark brown lesions were found on stems of some infected seedlings but none of the infected seedlings were killed. Also, no symptoms were found on mature leaves. The pathogen produced numerous sporangia on sporangiophores on diseased leaves under humid conditions. The disease on young seedlings was observed again in another litchi orchard at Caotun, Nantou County, in 2003. P. litchii was consistently isolated from diseased leaves. Two isolates from colonized longan seedlings, Tari 20250 collected in 2000 and Tari 23301 collected in 2003, were used for further studies. Both isolates produced large numbers of sporangia on long sporangiophores when cultured on 5% V8 agar (5% V8 juice, 0.02% CaCO3, and 1.5% agar). Sporangia produced on the same sporangiophores matured almost simultaneously. Sporangiophores 240 to 1,600 µm (mean 623 µm) branched dichotomously two to eight times. Sporangia were oval or lemon-shaped with semispherical papilla and deciduous with very short pedicels (2 to 5 µm). The dimension was 25 to 55 (35.25) × 15 to 27.5 (21.2) µm for sporangia and 0.5 to 1 (0.55) µm for pedicels. The length/breadth (L/B) ratio of sporangia was 1.3 to 2.14 (1.67). Both isolates produced numerous oospores on 5% V8 agar cultures in darkness. Artificial inoculation tests were done by spraying 5 mL of sporangial suspension (1,000 sporangia/mL) on each longan seedling without wounding. Results showed that both longan isolates of P. litchii were pathogenic on young longan seedlings, causing symptoms similar to those observed on leaves and stems of naturally infected longan seedlings in litchi orchards. Also, both longan isolates of P. litchii caused downy blight on fruits of litchi (L. chinensis var black leaf) by artificial inoculation tests. Moreover, a P. litchi isolate from litchi caused symptoms of leaf blight on young longan seedlings. P. litchii was reisolated from the infected longan tissues. The ribosomal internal transcribed spacer (ITS) sequence confirmed that the longan isolate Tari 20250 (GenBank Accession No. JQ814693) was 100% identical to other P. litchii isolates (GenBank Accession Nos. Gu111613 to Gu111615). To our knowledge, this is the first report of longan as a natural host of P. litchii. The study also suggests that P. litchii on volunteer longan seedlings in litchi orchards may be a potential source of inoculum for fruit downy blight of litchi. References: (1) C. C. Chen. Special Publ. Coll. Agric., Natl. Taiwan Univ. 10:1, 1961. (2) W. H. Ko et al. Mycologia 70:380, 1978.

First Report of Phytophthora cambivora Causing Leaf and Stem Blight and Root Rot on Taiwan Cherry (Prunus campanulata) in Taiwan.

Taiwan cherry or Formosan cherry (Prunus campanulata Maxim.) is a beautiful ornamental tree that is native to Taiwan. In spring 2005, a severe disease was observed on 1- to 3-year-old seedlings of Taiwan cherry in a garden in Tungshih, Taichung, Taiwan. Infected plants showed symptoms of greenish water-soaked spots on leaves that became dark brown, 2 to 3 cm in diameter. Infected leaves withered and fell to the ground in 3 to 5 days and young shoots showed symptoms of withering and drooping. Infected roots showed symptoms of necrosis. Severely infected plants eventually died. A Phytophthora sp. was isolated consistently from diseased samples of Taiwan cherry and associated soil. Six isolates of Phytophthora, of the A1 mating type (1), were isolated from single zoospores. Two of these isolates, Tari 25141 (deposited as BCRC34932 in Bioresource Collection and Research Center, Shinchu, Taiwan) and Tari 25144 (BCRC34933), were used for pathogenicity tests on 1-year-old seedlings of Taiwan cherry to fulfill Koch's postulates. Inoculation was done by placing a cotton swab containing zoospore suspension on leaves or stem, or by soaking seedlings in the zoospore suspension. Inoculated seedlings were kept in a greenhouse at 20 to 25°C for 30 days and examined for appearance of symptoms. Results showed that both isolates were pathogenic on seedlings of Taiwan cherry, causing symptoms similar to those observed on naturally infected seedlings. The temperature range for growth of the six isolates of Phytophthora was 8 to 32°C with optimum temperature at 24°C. The linear growth rate was 72 mm per day on V8A culture (5% V8 vegetable juice, 0.02% CaCO3, and 2% Bacto agar) at 24°C. The colonies on potato dextrose agar produced sparse aerial mycelia with conspicuous radiate patterns. Sporangia were sparse on V8A agar blocks, but abundant when the agar blocks were placed in water under continuous white fluorescent light (average 2,000 lux) for 2 days. Sporangiophores branched sympodially. Sporangia were pear shaped, nonpapillate and nondeciduous, 50 to 75 (62) × 30 to 48 (40) µm, with a length/width ratio of 1.2 to 2.2 (1.6). New internal nested proliferate sporangia were formed inside the empty sac of old matured sporangia after releasing zoospores. No chlamydospores were formed on V8A. Hyphal swellings with distinctive irregular catenulation were produced on V8A and in water. The pathogen was stimulated to form its own oospores by the A2 tester using the method described by Ko (1). Oogonia were 28 to 50 (40) µm in diameter with smooth or irregularly protuberant walls. Oospores were mostly aplerotic and 18 to 42 (31) µm in diameter. Antheridia were amphigynous, mostly two-celled, and 10 to 42 (29) × 12 to 24 (19) µm. The sequence of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA of isolate Tari 25141 (GenBank Accession No. GU111589) was 831 bp and had 99% sequence identity with a number of Phytophthora cambivora isolates such as GenBank Accession Nos. HM004220 (2), AY787030, and EF486692. Based on the morphological characteristics of sporangia and sexual structures and the molecular analysis of ITS sequences, the pathogen from Taiwan cherry was identified as P. cambivora (Petri) Buis. To our knowledge, this is the first report of P. cambivora on native Taiwan cherry in Taiwan and, so far, no other natural hosts have been reported. References: (1) W. H. Ko. J. Gen. Microbiol. 116:459, 1980. (2) P. W. Reeser et al. Mycologia 103:225, 2011.

Lasiodiplodia theobromae as a Causal Agent of Kumquat Dieback in Taiwan.

Kumquat (Fortunella margarita (Lour.) Swingle) is an important citrus fruit crop of Iland County in northeastern Taiwan. Fruit produced in this area are mainly for making preserves, which is a well-known product in Taiwan. Decline of kumquat was first noticed in 1990. Since 1995, it has become an important problem affecting ≈80% of kumquat orchards. The problem was especially serious after the passage of a typhoon. Some orchards were abandoned due to death of many declining trees. Initial symptoms were yellowing and browning of leaves on some branches of affected trees. Abscission of leaves and fruits occurred, subsequently resulting in the appearance of dieback of affected branches. Disease symptoms could progress to other branches of the same tree, gummosis on the trunk, and eventually death of the tree. Previously, Phytophthora citrophthora (Smith & Smith) Leonian was reported to be a causal organism of kumquat decline (1). Recently, isolations from declining kumquat trees in several orchards failed to recover P. citrophthora and instead we isolated Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (syn. Botryodiplodia theobromae Pat.) from tissue taken from the margin of discolored bark and wood on symptomatic branches. The fungus produced grayish black colonies on V8 agar and black ostiolate pycnidia (125 to 650 µm in diameter) with ovoid to elongate conidia (20 to 32 × 12 to 16 µm) on autoclaved whole wheat grains that were placed on V8 agar. Young conidia were hyaline and nonseptate, whereas mature conidia were brown, one septate, and striate. Pathogenicity tests were carried out on healthy kumquat trees located at the Taiwan Agricultural Research Institute experimental farm. Three isolates of L. theobromae were cultured on wheat-oat medium. Colonized grains (≈5 g) were held against a wound made by lightly scrapping a branch (5 to 7 mm in diameter) 200 to 250 cm above the ground by wrapping a sheet of clear plastic around the branch (2). Eighteen branches were used for each isolate, and the same number of branches similarly inoculated with sterile grains as a control. Healthy branches inoculated with L. theobromae showed disease symptoms similar to those observed on naturally infected plants. Discoloration of leaves on inoculated branches occurred within 1 week. Subsequently, all the leaves fell and infected branches died. The numbers of inoculated branches killed by the three isolates of L. theobromae tested were 18, 9, and 14 after 1 month. All control branches remained disease free. L. theobromae was reisolated from symptomatic tissues, completing Koch's postulates. The same isolates were used to inoculate trunks of kumquat trees with the method described above. Six trunks were inoculated with each isolate, and the same number of trunks similarly inoculated with sterile grains was used as the control. Gummosis on inoculated trunks occurred in 1 week. The numbers of inoculated trunks showing gummosis induced by the three isolates of L. theobromae tested were 6, 5, and 6 after 1 month. L. theobromae was reisolated from symptomatic tissue. All control trunks remained free of gummosis. Our results show that in addition to P. citrophthora, L. theobromae can also cause a dieback on kumquat. To our knowledge, this is the first report of disease caused by L. theobromae on kumquat or on any species in the Rutaceae family in Taiwan. References: (1) P. J. Ann et al. Plant Pathol. Bull. (Taiwan) 6:198, 1997. (2) W. H. Ko et al. Plant Pathol. 35:254, 1986.

Root and Foot Rot of Loquat in Taiwan Caused by Phytophthora.

Loquat trees growing in central Taiwan were inflicted with a disease causing wilting and death of plants due to severe foot and root rot. The vascular tissues of all infected plants turned brown. Typical as well as atypical isolates of Phytophthora parasitica were isolated from the diseased basal stem and root tissues but not from the discolored vascular tissues. Symptoms observed in the field were reproduced when roots and stems of loquat seedlings were inoculated with zoospores of atypical isolates of P. parasitica; whereas only fibrous root rot resulted from inoculation with typical isolates of P. parasitica. Atypical isolates could be differentiated from the typical isolates of P. parasitica by several characteristics, including colony appearance, partial caducous sporangia, size of oospore, growth at 36°C, mycelial soluble protein patterns, and pathogenicity.

Brown Root Rot of 10 Species of Fruit Trees Caused by Phellinus noxius in Taiwan.

Wilt and death of fruit trees was surveyed from 1990 to 1996. Most trees with decline symptoms in central and southern Taiwan had brown root rot caused by Phellinus noxius. The infected trees included litchi, sugar-apple, plumum, pear, loquat, persimmon, carambola, wax apple, grape, and jellyfig, with ages ranging from young seedlings up to 10-year-old trees. Symptoms were leaf discoloration, unthrifty appearance, and eventual death. Upon inoculation of the roots with P. noxius grown in a wheat-oat medium, healthy seedlings of these 10 tree species were killed. P. noxius was reisolated from diseased tissues of inoculated roots. The isolates of P. noxius obtained from different hosts were similar in morphological characteristics. Host specificity among the tested isolates was not observed in the cross-inoculation test, but variability in virulence was noted in some tests. Mycelial growth of different isolates of P. noxius showed similar temperature response, but the growth rates varied dramatically. Fruiting bodies were rarely found in the field. However, the fungus produced fruiting bodies artificially, on a sawdust medium, that were similar to those produced on the lower stems of declining trees. Brown root rot can be induced by replanting seedlings in infested soil or by contact with the diseased or dead roots. P. noxius causing brown root rot of these 10 fruit trees has not been previously reported.

Thin-walled microvessels in human coronary atherosclerotic plaques show incomplete endothelial junctions relevance of compromised structural integrity for intraplaque microvascular leakage.

OBJECTIVES: This study sought to examine the ultrastructure of microvessels in normal and atherosclerotic coronary arteries and its association with plaque phenotype. BACKGROUND: Microvessels in atherosclerotic plaques are an entry point for inflammatory and red blood cells; yet, there are limited data on the ultrastructural integrity of microvessels in human atherosclerosis. METHODS: Microvessel density (MVD) and ultrastructural morphology were determined in the adventitia, intima-media border, and atherosclerotic plaque of 28 coronary arteries using immunohistochemistry for endothelial cells (Ulex europeaus, CD31/CD34), basement membrane (laminin, collagen IV), and mural cells (desmin, alpha-smooth muscle [SM] actin, smoothelin, SM1, SM2, SMemb). Ultrastructural characterization of microvessel morphology was performed by electron microscopy. RESULTS: The MVD was increased in advanced plaques compared with early plaques, which correlated with lesion morphology. Adventitial MVD was higher than intraplaque MVD in normal arteries and early plaques, but adventitial and intraplaque MVD were similar in advanced plaques. Although microvessel basement membranes were intact, the percentage of thin-walled microvessels was similarly low in normal and atherosclerotic adventitia, in the adventitia and the plaque, and in all plaque types. Intraplaque microvascular endothelial cells (ECs) were abnormal, with membrane blebs, intracytoplasmic vacuoles, open EC-EC junctions, and basement membrane detachment. Leukocyte infiltration was frequently observed by electron microscopy, and confirmed by CD45RO and CD68 immunohistochemistry. CONCLUSIONS: The MVD was associated with coronary plaque progression and morphology. Microvessels were thin-walled in normal and atherosclerotic arteries, and the compromised structural integrity of microvascular endothelium may explain the microvascular leakage responsible for intraplaque hemorrhage in advanced human coronary atherosclerosis.

Vasculin, a novel vascular protein differentially expressed in human atherogenesis.

Recent suppressive subtractive hybridization analysis on human atherosclerotic plaque-derived RNA revealed genes upregulated in plaques with a thrombus versus stable plaques. Clone SSH6, containing part of a putative open reading frame of an unknown protein, was further investigated. Full-length cDNA, coding for a 473-amino acid (aa) protein, was identified in a vascular smooth muscle cell (SMC) cDNA library. Bioinformatics suggested the presence of multiple SSH6 variants due to alternative splicing of exon 3. Multiple-tissue Northern blot analysis demonstrated a differential expression pattern of these variants, as a ubiquitously expressed SSH6 mRNA missing exon 3, was detected apart from a putative vascular SMC-specific form containing exon 3. Western blot analysis indicated a ubiquitous 35-kDa protein (SSH6-beta), in addition to a 45-kDa protein (vasculin), detected in the vascular wall and in plasma. Analysis of arteries displaying various stages of atherosclerosis indicated that the vasculin/SSH6-beta ratio increases throughout atherogenesis. Immunohistochemical analysis demonstrated cytoplasmic expression of SSH6 gene products in macrophages, endothelial cells, and SMCs. In summary, we identified a novel mRNA/protein, vasculin, in the arterial wall and plasma. The regulated expression of vasculin in plaques suggests a role in atherogenesis. Moreover, its presence in plasma opens perspectives for vasculin as a marker for atherosclerosis.