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College of Pastoral Agriculture Science and Technology, Lanzhou University; Gansu Grassland Ecological, Research Institute, P.O. Box 61, Lanzhou 730020, China
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ABSTRACT |
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A new species, Embellisia astragali sp. nov., is described from necrotic leaves, petioles and stems of Astragalus adsurgens in China. The morphology of E. astragali is compared and contrasted to that of four similar species, E. abundans, E. oxytropis, E. phragmospora and E. telluster. The fungus grew intercellularly in stems and leaf blades and intracellularly in leaves. It was isolated from most sources of seeds at frequencies of 0.1–44.6%. Growth rates of colonies on potato-carrot agar, potato-dextrose agar, wheat hay decoction (WHDA) and V8 at 25 C were 0.48, 0.32, 0.68 and 0.27 mm d–1, respectively. The optimal temperature for colony growth on WHDA was 20–25 C, and no growth was measured above 30 C. Five week old standing milk-vetch seedlings were inoculated with E. astragali by dipping whole roots and pruned roots in a conidial suspension and pouring the suspension onto the soil surface in which two seedlings had been planted. After 20 wk 66.5%, 62.1% and 85.0% plants were diseased and 24.1% , 20.7% and 17.5% plants were dead, respectively. Symptoms included the development of more side shoots with small, curved, necrotic and yellowed young leaves, plant stunting, reddish brown lesions, stem browning, dieback, shoot blight, crown rot, root black rot and plant death. This is first report of a pathogenic Embellisia on legumes.
Key words: brown stem, growth rate, leaf lesion, pathogenicity, root rot
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTS AND DISCUSSIONLITERATURE CIED |
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, Roalson and Allred 1997, Laguerre et al 1997, Wojciechowski 2005). It has been cultivated widely in arid areas of northern China as forage. Moreover it is used as a green manure rotation crop and as a windbreak or cover plant to protect soil from erosion (Fu et al 1993).
In recent years a new and devastating disease of standing milk-vetch has been observed in the Huan County Grassland Experimental Station (37°7’N, 106°49’E, 1650 m elevation) in Gansu Province, China, resulting in seriously degraded milk-vetch pastures. This station is in a typical temperate, semi-arid monsoon climate where annual mean air temperature was 7.1 C, annual precipitation was 359.3 mm and annual evaporation was 1993.3 mm. A fungus was isolated regularly from infected plants and determined to be a species of Embellisia Simmons (1971, 1983).
Embellisia is recognized on the basis of a combination of characters including predominantly transversely septate conidia with distinctly thickened and pigmented septa, variously swollen, curved or sigmoid conidia, umbilicate sites of conidium production at conidiophore geniculations and intrahyphal proliferating chlamydospores in culture (Simmons 1971). Although no one character is firmly diagnostic, the comparatively thick and dark conidial septum remains the best character to differentiate Embellisia from similar genera (Simmons 1986). We determined that this is an undescribed species based on comparison of morphology as well as biological characteristics and pathogenicity of described species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTS AND DISCUSSIONLITERATURE CIED |
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, then aseptically cut into 5 mm segments. Ten segments per tissue of each plant were placed on the surface of 2% potato-dextrose agar (PDA) and incubated at 22 C for 5 d. Morphologically distinct colonies growing from cut ends or segments were transferred to PDA. Frequency of isolation of one slow-growing fungus was determined, and this fungus subsequently was studied in depth. Isolation from seeds.— – Standing milk-vetch seeds harvested in the field in 2003 and 2004 from diseased plants and in 2005 from diseased and healthy plants from Liaoning Province, Shaanxi Province, Ningxia Automatic Region and Inner Mongolia Automatic Region, were surface disinfected as above, then plated on PDA and incubated at 25 C for 7 d. Five hundred seeds of each source were plated, except only 50 were available and plated from the Huan County seeds harvested in 2003. Twenty-five seeds were placed in each Petri dish (90 cm diam). Frequency of isolation of the slow-growing fungus was recorded for each seed source.
Morphology.— –
The description of Embellisia astragali is based on an isolate (LYZ 1412) from a brown stem of standing milk-vetch growing in the experimental station. Colonies of the fungus developed slowly on PDA so mycelium was transferred onto wheat hay decoction agar (WHDA, dry wheat hay 20 g, agar 20 g, distilled water 1000 mL, wheat hay was decocted for 20 min) and incubated at 25 C for 4 wk. Disks then were made with a 4.5 mm cork borer from the margins of colonies, and one disk was transferred onto potato-carrot agar (PCA), PDA, WHDA and V8 agar plates (90 mm diam). The plates were sealed with film, incubated at 22 C in 16/8 h fluorescent light/dark illumination cycle as described by Simmons (1971). Colony characteristics were observed weekly with the naked eye and a dissecting microscope (Leica MS5, 50x), and sporulating characteristics and morphologies were observed under a light microscope (Olympus, CH, 400x and 1000x). Conidiophores and conidia produced on PCA (agar, Damao, Tianjin Damao Chemical Reagent Co. Ltd.) were mounted in tap water and measured at 400x. Sizes are based on 50 measurements of each character.
Dead plants covered by a layer of thick, black mold also were collected from the station in Aug 2004, and conidia and conidiophores produced on those plants were observed and measured. Pure cultures were obtained by isolating single spores from the dead plants.
Collections were deposited in the Mycological Herbarium of the Lanzhou University (MHLZU), Lanzhou, China. A dried culture of isolate LYZ 1412 from a brown stem is designated the holotype of Embellisia astragali as MHLZU 0408.
Biological characteristics.— – Growth rate of the isolate LYZ 1412 cultured on PCA, PDA, WHDA and V8 at 25 C in the dark were determined by measuring colony diameters after 4 wk. To determine the effect of temperature plates of WHDA were inoculated and incubated at 5, 10, 15, 20, 25, 30 and 35 C in the dark for 4 wk. Each treatment had four replicates and all plates were sealed with film.
Observation of conidial germination.— – Germination of conidia obtained from the surface of dead plants and isolated from infected plants was observed after individual conidia were incubated in sterile distilled water for 48 h at 22 C.
Examination of hyphae in plant tissues.— – Young stems and leaf blades of standing milk-vetch were excised from inoculated and uninoculated plants 7–20 wk postinoculation (plants were 12–25 wk old) and from healthy and naturally infected plants collected from the field May–Aug 2004. Stems were longitudinally and transversely hand sectioned. Leaf blades were soaked in pyridine 24 h then stained with aniline blue and sectioned into 1 mm2 pieces. All sections were observed under a light microscope. The remains of tissues after examination were plated on PDA to determine the fungal species growing within plants. Simple examination methods frequently were applied for determining relationship between symptom expression and hyphal development within plants and stems and leaf blades collected in the field and in inoculation experiments. Small pieces of leaf blades were placed on a slide, squeezed and observed under a microscope. Stems were sectioned and only pith of stems was observed under a microscope without staining.
Pathogenicity.— – Pathogenicity testing was carried out in a 25 C air-conditioned greenhouse with 14 h light/d. Seeds of standing milk-vetch used in inoculations were collected from Liaoning Province and were free of the fungus based on attempted isolations as described in seed isolation section above. Surface-disinfected seeds were pregerminated on moist filter paper for 4 d and transplanted into soil sterilized at 169 C for 5 h. The 105 conidia mL–1 inoculum of E. astragali (LYZ 1412) was prepared from 2 mo old WHDA cultures. Five week old seedlings were inoculated (i) by removing the seedlings and dipping roots in a conidial suspension for 30 min as described by van Steekelenburg (1980), (ii) as above but pruning 1 cm off of root tips before dipping and (iii) by pouring 5 mL of the conidial suspension onto the soil surface in each paper cup as described by van Steekelenburg (1980). For the root-dip and pruned-root dip inoculation, inoculated seedlings were placed in fresh sterilized soil. Seedlings treated with sterile distilled water served as controls for each type of inoculation. Two seedlings were grown in each cup and watered daily. A total of 16 paper cups (7 cm diam) were used for each treatment and for each control and arranged in a randomized design. Twenty weeks after inoculation the number of infected and dead plants was recorded and all plants were removed and symptoms on roots were examined. Portion of leaves, stems and roots of inoculated and uninoculated plants from each treatment were placed on PDA and incubated 7 d to re-isolate the fungus.
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TAXONOMY |
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–12
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HOLOTYPUS. – Cultura exsiccate in agaro ex LYZ 1412, in Astragalus adsurgens Pall., Huanxianensis, Gansuensi Provincia, Sinico, 25 viii 2004, Y. Z. Li, MHLZU 0408.
Colonies on PCA white, gray to dark brown, sometimes olivaceous on the margins, with aerial lanose mycelium, reverse white to dark brown (FIG. 2
). Submerged hyphae radial, arborescent, swollen and branched. Chlamydospores abundant, developing as chains of reddish brown, inflated, thick-walled, septate cells.
Primary conidiophores 0–1-septate and short or several septate and elongate, from branched aerial hyphae or hyphal tips that grow erectly upward from the agar substrate, simple or branched, geniculate with 2–5 conidiogenous loci, pale olivaceous to medium golden brown, 94–197 x 3–5 µm (mean 130 x 4 µm). Both primary and secondary sporulation relatively sparse. Conidia predominantly long obclavate, straight or Y-shaped, slightly inequilateral to conspicuously curved and even sigmoid, basal cells nearly sphaeroid, 3–6(–8) transverse septa, and no or rarely 1 longitudinal/oblique septum, distinctly constricted at the septa, 24–66 x 8–13 µm (mean 45 x 11 µm). Young conidia pale olivaceous and semi-transparent, mature conidia medium golden brown with thick, dark septa, smooth-walled. Secondary conidiophores apical, basal, sometimes lateral from primary conidia, 1–2-celled, pale, tip-swollen or elongate, 2–3-geniculate at conidiogenous loci, rarely branched. Conidial chains fewer than three units (FIG. 6). Broadly holoblastic conidioid elements present. Hyphal coils not observed. Teleomorph unknown.
Conidiophores emerging from the surface of dead infected plants, clustered or scattered, conidiogenous loci with white center and brown margin, 29–129 x 5–8 µm (mean 66 x 6 µm) (FIG. 8). Most conidia with 6–8(–11) transverse septa, 32–71 x 8–13 µm (mean 57 x 11 µm) (FIG. 9). Other morphologies were same as those on PCA.
Aerial hyphae on WHDA (FIG. 3) sparse, and submerged hyphae abundant, forming a layer of "black mold" around agar consisting of conidiophores and conidia. Colonies on PDA (FIG. 4) and V8 agar (FIG. 5), white to black, abundant, elevated aerial hyphae around margins, sparse aerial hyphae at centers. Conidial colors, shapes, sizes and septa on PDA were same as those on PCA.
Specimen examined. – CHINA, GANSU PROVINCE: Huan County Grassland Experimental Station, 37°7’N, 106°49’E, isolated from a brown stem of standing milk-vetch (Astragalus adsurgens Pall.), 25 Aug 2004, Y. Z. Li (HOLOTYPE: MHLZU 0408; EX-TYPE: LYZ1412); Huan County Grassland Experimental Station, 37°7’N, 106°49’E, Gansu, China, on the surface of a dead black stem of standing milk-vetch (Astragalus adsurgens Pall.), 25Aug 2004, Y.Z. Li (MHLZU 0409; living culture LYZ 1416).
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RESULTS AND DISCUSSION |
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Isolation from seeds.— – E. astragali was isolated from seeds from most sources except Liaoning. Frequencies of isolation of the fungus from seeds collected from Shanxi, Ningxia and Inner Mongolia were 0.1%, 0.2% and 1%, respectively, and that from Huan County seeds harvested in 2003, 2004 and 2005 were 22%, 44.6% and 6%, respectively. These results suggest that E. astragali is seed-borne and the disease caused by the fungus occurs in most provinces of northern China.
Frequencies of isolation from most sources of seeds were comparatively low, possibly because most of the diseased shoots do not blossom in the field or because most of the infected seeds are brown and shriveled and therefore would be eliminated from seed production.
Morphology.— – Embellisia astragali is similar to E. abundans, E. oxytropis, E. phragmospora and E. telluster, which also have obclavate or long ellipsoid conidia with no or 1–2 longitudinal septa. However the conida of E. astragali are different from the others in being long obclavate, larger, markedly constricted at the septa, with nearly sphaeroid basal cells. E. astragali is most similar to E. oxytropis when both were cultured on PDA, but conidia of E. astragali are long obclavate with 3–6(–8) transverse septa, medium golden brown, with no or rarely 1 longitudinal/oblique septa; secondary conidiophores can be produced from any conidial cell whereas conidia of E. oxytropis are long ellipsoid or cylindroid, with (2–)3–4 transverse septa and no longitudinal/ oblique septa, dark brown, and secondary conidiophores are produced from the apex of primary conidia. Furthermore colonies of E. astragali can continue radial growth at 25 C and become white to black, which differs from E. oxytropis. Although both grow within host plants, E. astragali is a pathogen of A. adsurgens while E. oxytropis is an endophyte of Oxytropis kansuensis.
Biological characteristics.— – E. astragali grew at 5–25 C on WHDA but not above 30 C. The optimal temperature for colony growth was 20–25 C. Growth on PCA, PDA, WHDA and V8 were 0.52, 0.34, 0.63 and 0.34 mm d–1 at 25 C in the dark, respectively.
Observation of hyphae in plant tissues.— –
Hyphae of E. astragali (1–5 µm diam) were observed in symptomatic stems and leaf blades of naturally infected plants in the field and of artificially inoculated plants, but they never were observed in the uninoculated plants 20 wk after inoculations and asymptomatic plants grown in the field in Aug 2004. However they sometimes were observed in the asymptomatic tissues of plants grown in the field in May and Jun 2004 and in inoculated plants 7–12 wk after inoculations. This fungus developed intercellularly between parenchyma cells in the pith of stems (FIGS. 10–11) and leaf blades (FIG. 12) and intracellularly under the epidermis of leaf blades (FIG. 12). When the upper leaf blades exhibited symptoms the hyphae were found in the pith of main stems regardless of whether stems were discolored. The fungal hyphae were determined to be E. astragali by the isolations from the remains of tissues after examination. With the simple examination method the hyphae were observed readily filling the pith of brown stems and necrotic leaf blades.
Conidial germination.— –
Conidia of E. astragali germinated from all cells (FIG. 7), which is consistent with the character of genus Embellisia (Simmons 1971). Germination was more frequent from the apical and basal cells of conidia of E. astragali and basal cells usually produced two germ tubes.
Pathogenicity.— – Symptoms exhibited at the 10th week postinoculation of pruned or unpruned dipping roots and at 7th week after pouring inoculum onto soil surface initially were observed on young lateral shoots or on main stems or on the upper leaf blades of inoculated plants. These symptoms included development of side shoots with small, curved, necrotic and yellowed young leaves, reddish brown lesions on petioles, stems browning, dieback, shoot blight, plant stunting, crown rot, root black rot and plant death. Plants inoculated by root dip, pruned-root dip and pouring inoculum onto soil surface were 66.5%, 62.1% and 85% diseased, and 24.1%, 20.7% and 17.5% dead, respectively. All uninoculated plants remained healthy throughout the tests. E. astragali was re-isolated from symptomatic tissues of inoculated plants but was not isolated from healthy inoculated plants or uninoculated plants 20 wk postinoculation.
The results of the inoculation of root dip and cut-root dip indicate that E. astragali initially invades roots then expands to above-ground parts of standing milk-vetch. When inoculated by dispersing the co-nidial suspension onto the soil surface, the fungus probably invades the stem base then moves into the roots and leaves. If the fungus grew into the soil then infected the roots and moved upward the incubation period would be longer than that of dipping-root inoculations. Moreover crown rot and stem browning in this inoculation were more serious and roots were less damaged than in the other types of inoculations.
To date 22 species of Embellisia have been described (Simmons 1971, 1983, 1990, 2004, de Hoog and Muller 1973, Mutanjola-Cvetkovi
and Ristanovi 1976, de Hoog et al 1985, David et al 2000, Wang et al 2006). Three Embellisia species, E. astragali, E. eureka (Simmons 1990) and E. oxytropis (Wang et al 2006), are associated with legumes but E. astragali is the only species pathogenic to its host. Diseases caused by other species of Embellisia include yellow leaf lesions of Brussels sprouts (Brassica oleracea L. var. gemmifera) caused by E. abundans (Lumyong et al 1984), brown lesion of a Pteridophyte caused by E. phragmospora (van Emden 1970, Cao et al 1990), bulb canker of garlic (Allium sativum) caused by E. allii (Simmons 1971) and skin spot of bulb and leaf spot of Hyacinthus orientalis, Freesia refracta, Scilla sibirica and Muscari sp. caused by E. hyacinthi (de Hoog and Muller 1973, David 1991). The disease of standing milk-vetch caused by E. astragali is devastating to field production of the crop and seems to be one of the most serious caused by species in this genus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author E-mail: zhibiao{at}lzu.edu.cn
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LITERATURE CIED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTS AND DISCUSSIONLITERATURE CIED |
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