Vanda Orchid Research Articles

Detection, diagnosis of orchid virus and inactivation of cymbidium mosaic virus (CYMV) on plants

Floriculture is one of the disciplines of Horticulture which is dealing with growing of ornamental plants flowering plants and garden maintenance etc. orchids are one of the floriculture plant. It is a member of Orchidaceae family consisting of more than 25,000 species, which are distributed almost all over the world. Vanda Orchid plant is collected from different nurseries showing chlorotic and mosaic symptoms were observed and it was suspected to infect with virus. So the symptomatic plants were tested with Direct Antigen Coating- Enzyme Linked Immunosorbent Assay (DAC-ELISA) for Cymbidium Mosaic Virus (CYMV), Odontoglossum ring spot virus (ORSV), Poty virus and Tomato Spotted Wilt Virus (TSWV) and further confirmed by Transmission Electron Microscopy (TEM). With the two methods CYMV were detected positively from the samples and low positive results were observed for ORSV, Potex, Poty virus and Tomato Spotted Wilt Virus (TSWV). High incidence of Cymbidium Mosaic Virus (CYMV) was observed. Chemicals were evaluated for inactivation of CYMV on pruning tools for disease control. Skimmed milk was found to be effective, non caustic and inexpensive for inactivation of CYMV inoculated on local lesion indicator host plants. However, systemic host plants (Orchids) were used in evaluation.

Effect of potato extract on in vitro seed germination and seedling growth of local Vanda roxburgii orchid

An experiment was conducted to investigate the effect of potato extract (PE) on seed germination and seedling growth of Vanda roxburgii in vitro, a local orchid. Potato extracts significantly enhanced seed germination and seedling growth. Among the concentrations, PE at 200 mlL-1 enhanced per cent germination from 17.2% (control) to 78.24% and was found to be the most suitable one for seed germination. It was also the best among treatments for production of viable protocorms as well as healthy seedlings from seeds. Growth of seedlings in respect of length of shoot, number of root per shoot and length of root per seedling was significantly enhanced in medium supplemented with 100 mlL-1 of PE. It may be concluded that PE at optimum concentration could be supplemented in medium for high percentage of seed germination and seedling growth of Vanda orchid. DOI: http://dx.doi.org/10.3329/jbau.v9i2.10988 J. Bangladesh Agril. Univ. 9(2): 211–215, 2011

First Report of Sclerotium rolfsii on Ascocentrum and Ascocenda Orchids in Florida.

Southern blight caused by Sclerotium rolfsii is known to occur on several economically important orchid hosts, including Vanda species and hybrids (1-3). In the summer and fall of 2008, an outbreak of southern blight on Vanda orchids was seen in several commercial nurseries and landscapes throughout South Florida. More than a dozen orchids were affected at one of the locations, and symptoms of S. rolfsii were observed on Ascocentrum and Ascocenda orchids, which are also common in the trade and demand a resale value ranging from $20 to $150 for specimens in bloom. Affected Ascocentrum and Ascocenda orchids were found severely wilted at the apex, while around the base of the plants, tan, soft, water-soaked lesions were present. As the lesions progressed, leaves around the base of the plants began to fall off, leaving the stems bare. After 2 days, white, flabellate mycelium was seen progressing up the stem and numerous, tan-to-brown sclerotia were present. Leaves and portions of the stems were plated on acidified potato dextrose agar (APDA) and grown at 25°C. White, flabellate mycelium and tan sclerotia approximately 2 mm in diameter were produced in culture and microscopic examination revealed the presence of clamp connections. The fungus was identified as S. rolfsii and a voucher specimen was deposited with the ATCC. A PCR was performed on the ITS1, 5.8S rDNA, and ITS2 and the sequence was deposited in GenBank (Accession No. GQ358518). Pathogenicity of an isolate was tested by placing 6-mm plugs taken from APDA plates directly against the stem of five different Ascocentrum and Ascocenda orchids. Five Ascocentrum and Ascocenda orchids were inoculated with 6-mm plugs of plain APDA and five were untreated controls. Plants were housed under 50% shade, 60 to 95% humidity, and temperatures ranging from 75 to 88°F. Within 7 days, all inoculated plants developed symptoms that were identical to those observed on original plants and S. rolfsii was consistently reisolated from symptomatic tissue. Ascocentrum and Ascocenda were previously reported under miscellaneous orchid species and hybrids as hosts for S. rolfsii (1). However, this report was highly ambiguous and the most current edition does not report the host fungus combination (2). To our knowledge, this is the first report of S. rolfsii affecting Ascocentrum and Ascocenda orchids.

First Report of Bacterial Soft Rot on Vanda Orchids Caused by Dickeya chrysanthemi (Erwinia chrysanthemi) in the United States.

Vanda orchids are epiphytes grown for their attractive flowers by commercial producers and hobbyists throughout Florida. In August 2007, five Vanda hybrids, with an economic value of $150 each, were found at a nursery in central Florida with leaves that were macerated, brown, and water soaked. According to the growers, the plants were normal the previous day but symptoms developed rapidly. The plants were immediately removed from the greenhouse to prevent potential disease spread. Bacteria were isolated according to the method of Schaad et al. (1). Isolated bacteria grew at 37°C, were gram negative, degraded pectate, and produced phosphatase. MIDI (Sherlock version TSBA 4.10; Microbial Identification 16 System, Newark, DE) (SIM 0.906) identified the bacteria as Erwinia chrysanthemi (Dickeya chrysanthemi Burkholder et al. 1953) Samson et al. 2005. PCR was performed on the 16S rRNA gene (GenBank Accession No. EU526397) with primers 27f (5'-GAGAGTTTGATCCTG GCTCAG-3') and 1495r (5'-TACGGCTACCTTGTTACGA-3') (2). Subsequent DNA sequencing and GenBank search showed the isolated strain is 99% identical to that of Dickeya chrysanthemi. Four leaves each of six Vanda hybrids were inoculated by injecting approximately 150 μl of a bacteria suspension at 1 × 108 CFU/ml into each leaf. One plant was inoculated with water in each of four leaves. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 29°C day and 17°C night temperatures. Within 24 h, soft rot symptoms appeared on inoculated leaves. The water control appeared normal. D. chrysanthemi was reisolated and identified with the above method, thus Koch's postulates were fulfilled. To our knowledge, this is the first report of a soft rot caused by D. chrysanthemi on Vanda hybrids. Because of the popularity and high value of Vanda orchids, proper identification of this rapidly progressing bacterial disease is of great importance for the commercial producer and homeowner alike.

Efficient production of transgenic plantls of Vanda through sonication-assisted Agrobacterium-mediated transformation of protocorm-like bodies

In Vanda orchids, it is important to produce cultivars with economically important traits such as disease and pest resistances and novel flower colors, which are difficult to achieve by conventional cross breeding methods. To realize these breeding objectives, it is now expected to apply genetic transformation technology to introduce useful foreign genes into Vanda orchids. However, there has been almost no information on the genetic transformation of Vanda. Transgenic plants were successfully regenerated after co-cultivating protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLBs of ‘Tokyo Blue’ maintained in liquid New Dogashima medium (NDM) under dark condition, were subjected to transformation experiments. The PLBs inoculated with Agrobacterium produced secondary PLBs 4 weeks after transfer onto 3 g l−1 gellan gum-solidified NDM containing 30 g l−1 maltose, 10 mg l−1 meropenem and 10 mg l−1 hygromycin. Transformation efficiency was increased by prolonged period of infection (240 min) and wounding treatment of PLBs by sonication (5 min) during inoculation. Transformation of the hygromycin-resistant plantlets regenerated from different PLBs was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization. With this transformation system, approximately 17 independent transgenic plants were obtained from 1 g PLBs.

Ethylene and Auxin Participation in Pollen Induced Fading of Vanda Orchid Blossoms

Blossoms fromii several varieties of orchids fade prematurely when their pollinia are removed or disturbed (2,3,7), and also if the flowers are gassed with ethylene (8, 9, 10), pollinated (3, /7), or treated with an auxin (3, 7). During natural Ifading as well as that induced by removal of the pollinia, ethylene is evolved (2, 3, 9, 10), andtherefore it has ibeen inferred that the gas is the catusative agent. Ethylene evolution also is stimlulated Nvllen plant tissuies are exposed to auXin ( 1, 4), andl wve now )resent evidence that this response is the basis for flower fading1 indtuced by pollination or an,xin application. Blossoms from ['undo Petanboeran or Van(la Rose .M1arie were iplaced wvitlh their cut ends in a beaker of water, and( sealed nudtler a 200 nlm bell jar having a ground glass base anid a side arm closed with a rubber vaccine cap. Otlher flowers svere pollinated, emilasculated (pollinia removed), self-pollinated, or treated with 5 mnm IAA in lanolin or 0.1 mm carboxyl lajbeled '-C-IAA in 0.8 % agar (specific activity 8 mc/mmole) applied to the stigmas, and then the flowers were sealed under a bell jar. Air samples, removed with a hlypodernmic syringe, were analyzed by gas chromatography to determine the ethylene content (4), anid at least every 10 hours the chambers were completely aerated. Ethylene evolution by isolated sepals and petals, columns or lips was measured by incubating each tissue in a 50 ml flask fitted with a vaccine cap. and sampling the air in the flask at hourly intervals. Cutting the floral parts induced a wound response, lasting about 1 hour, during whichi time 1.5 mkdJ of ethylene per gram of cut control tissue 'was produced. Treated cut tissue was only considered to produce ethylene if the total evolution exceeded this value, and if the extra evolution contintued at a nearlv constant rate for at least 4 hours. Intact blossoms were also placed in desiccators and gassed for 3 to 24 hours with 10 ppnm ethylene. In suich cases the flowNers were aerated f'or at least 1 hour 'before ethy;lenle production was measured in order to remiove any ethylene contained within the tissue. The sipread of 1AC-IAA from the stigmas of the flower was determined by excising the 'floral parts from representative blossomiis after 3, 6, and 24 hours and dividing these into pieces as illustrated in figure 3. Each piece was ground with ethanol on a planchet, dried, and counted w ith a 21 % efficient gas flow counter. When blossoms of V. Rose Marie were emasculated, ethylene evolution began after a 10 hour lag period (fig 1), and fading became obvious after an additional. 8 to 12 hours. A similar time course of ethvlene production and fadintg hias been reported for V. Miss Agnes Joaquitin blossoms after renoval of tlieir pollinia (2). whereas F. Petarnioeran, a semiterete of heav texture, fades after a considerablv longer lag(fig 2). That remiioval of the pollinia per se is not responsible for floral fading is indicated bv experiments with Phalcanopsis Pamiiala (7) which have showun that disturbance of the connection between the rostelluii (tthe structure supporting the pollinia) and the upper surface of the sticky disk with which it is in intimate contact, suffices to induce the response. There is no indication that this tvpe of fading ,in.volves a release or production of auxin, for it does not result in swelling of the column whereas both auxin application and pollination bring about such an effect. Self ,pollination induces ethylene formation within 1 hour in V. Rose Marie, and flower fading within 8 to 10 bhours (ifig 1). A simil.ar time course was observed with V. Petamboerant even when the flower was pollinated with its own pollinia intact (fig 2). This response was duplicated by applying enough IAA (5 mm) in lanolin paste to fill the stigmatic cavity (figo2), and exactlv the same result was obtained using 0.1 bM '*C-IAA in 0.8 % agar. Other cases .n which the fading response caused by pollination is stimulated by auxin have been reported (3, 7), and led to the suggestion that the large quantity df auxin contained in the pollen is responsible for the pollination effect (7). As auxin stimulates ethylene evolution in these blossoms and other plant tissues within an hour, an(d since ethylene causes floral fading, it is logical to conclude that this is the mlechanismll involved. Auxin induced ethylene formation must also influence the growvth of the column for within 2 hours after application of pollinia or auxin this structure