Improved attachment and parasitism of <Emphasis Type="Italic">Trichoderma</Emphasis> on <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> in vitro

Monoclonal and polyclonal antibodies that bind to eggs and/or second-stage juveniles of the nematode Meloidogyne javanica were tested for their effects on the parasitic interactions between this nematode and the fungus Trichoderma. Parasitism of Trichoderma asperellum-203 and Trichoderma atroviride on nematode egg masses, eggs and juveniles was enhanced when antibodies were incorporated into in vitro parasitism bioassays. Parasitism on separated eggs (without gelatinous matrix) and their hatched juveniles was also improved, compared to controls without antibodies that did not attach fungal conidia. Improved parasitism could be due to bilateral binding of the antibodies to the nematodes and conidia, enabling better conidial attachment to the nematodes. Enhanced germination of antibody-bound conidia further improved parasitism. Differences were observed among antibodies in their effects on fungal parasitism and their interaction with Trichoderma species. We focused mainly on the egg- and juvenile-binding monoclonal antibody MISC that exhibited a stronger reaction with T. asperellum-203 than with T. atroviride. Pretreatment of this antibody with fucose inhibited its binding to nematodes and conidial attachment to nematodes, as well as conidial agglutination in the presence of the antibody. Antibody binding to juveniles affected their movement and viability, especially gelatinous matrix-originated juveniles. The fucose-specific lectin Ulex europaeus-I enhanced conidial attachment to nematode life-stages, and conidial agglutination occurred in its presence. These phenomena were inhibited by preincubating lectin with fucose. Our results suggest that carbohydrate residues, such as fucose, on the surface of the nematode and fungal conidia are involved in the antibody- and lectin-mediated improved parasitism.     

Nematicidal potential of materials from <Emphasis Type="Italic">Medicago</Emphasis> spp.

The nematicidal effect of soil amendments with dry top and root material from Medicago sativa and/or Medicago arborea was evaluated on the root-knot nematode Meloidogyne incognita and on the cyst nematode Globodera rostochiensis in potting mixes. All amendments suppressed root and soil population densities of both nematode species compared to non-treated and chemical controls. The suppressiveness of M. sativa differed between top and root material and among the amendment rates. In field conditions soil amendments with 20 or 40 t ha⁻¹ of a pelleted M. sativa meal increased tomato crop yield and reduced soil population densities and root galling by M. incognita. It is suggested that saponins were at least partly responsible for the nematicidal activity.     

Evaluation of nematicidal properties of saponins from <Emphasis Type="Italic">Medicago</Emphasis> spp.

The nematicidal activity of saponins from Medicago arborea (tops), M. arabica (tops and roots) and M. sativa (tops and roots) against the plant-parasitic nematode Xiphinema index was investigated. Nematicidal activity of related prosapogenins and sapogenins on X. index is also described. Saponins from Medicago spp. at different concentrations were all nematicidal, those from M. arborea tops being the less effective. In general, saponins induced 100% mortality at 500 μg ml⁻¹ between 8 and 48 h, while prosapogenins resulted in toxicity starting at 125 μg ml⁻¹. Differences in the effects on X. index induced by prosapogenins and sapogenins were less pronounced, although prosapogenins displayed a larger range of activity. Assays with purified sapogenins demonstrated the relationship of the observed nematicidal activity of M. sativa and M. arborea to the content of the main aglycones (medicagenic acid and hederagenin, respectively) in the saponin extracts. Hederagenin displayed the highest bioactivity, giving 38% mortality after 1 h at 125 μg ml⁻¹.     

Susceptibility of eggs of <Emphasis Type="Italic">Tribolium confusum</Emphasis>, <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> and <Emphasis Type="Italic">Plodia interpunctella</Emphasis> to four essential oil vapors

Susceptibility of eggs of Tribolium confusum du Val. (Coleoptera: Tenebrionidae), Ephestia kuehniella (Zell.) (Lepidoptera: Phycitidae) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) to vapors of essential oil from garlic (Allium sativum L.), birch (Betula lenta L.), cinnamon (Cinnamonum zeylanicum (Blume)) and aniseed (Pimpinella anisum L.) was studied. Preliminary bioassay tests indicated that vapors of the essential oils had a significant effect on the eggs of tested insect species when exposed to a concentration of 20 μl l ⁻¹ air for 24 h. Generally, garlic and birch essential oils were more toxic to the eggs of tested insect species than cinnamon and aniseed essential oils (except for eggs of T. confusum). There was also a significant difference between susceptibility of eggs of T. confusum, E. kuehniella and P. interpunctella to tested essential oils. Toxicity data indicated that eggs of T. confusum were more susceptible to tested essential oils, with LC₉₀ values ranging from 3.11 to 33.49 μl l ⁻¹ air, than those of E. kuehniella and P. interpunctella; eggs of P. interpunctella were the most tolerant to the essential oils, with LC₉₀ values ranging from 22.02 to 72.42 μl l ⁻¹ air. Concentration × time (Ct) products of 0.29, 0.22, 0.13 and 1.37 mg h l ⁻¹ for garlic, birch, cinnamon and aniseed essential oil, respectively, were required to obtain 90% kill of T. confusum eggs. Although cinnamon essential oil had a much closer Ct product value to methyl bromide, garlic and birch essential oils were found to be the most promising ones since they had also high fumigant toxicity on eggs of both E. kuehniella and P. interpunctella.     

Resistance to <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> expresses a hypersensitive-like response in <Emphasis Type="Italic">Coffea arabica</Emphasis>

Root-knot nematodes (RKN) are obligate parasite species of the genus Meloidogyne that cause great losses in Arabica coffee (Coffea arabica L.) plantations. Identification of resistant genotypes would facilitate the improvement of coffee varieties aiming at an environmental friendly and costless nematode control. In this work, the C. arabica genotype ‘UFV 408-28’ was found to be resistant to the most destructive RKN species M. incognita. Pathogenicity assays indicated that the highly aggressive populations of M. incognita races 1, 2 and 3 were not able to successfully reproduce on ‘UFV 408-28’ roots and displayed a low gall index (GI = 2). An average reduction of 87% reduction of the M. incognita population was observed on ‘UFV 408-28’ when compared to the susceptible cultivar ‘IAC 15’. By contrast, ‘UFV 408-28’ was susceptible to the related species M. exigua and M. paranaensis (GI = 5 and 4, respectively). Histological observations performed on sections of UFV408-28 roots infected with M. incognita race 1 showed that nematode infection could be blocked right after penetration or during migration and establishment stages, at 6 days, 7 days and 8 days after infection (DAI). Fluorescence and bright field microscopy observations showed that root cells surrounding the nematodes exhibited HR-like features such as accumulation of phenolic compounds and a necrotic cell aspect. In the susceptible ‘IAC 15’ roots, 6 DAI, feeding sites contained giant cells with a dense cytoplasm. Necrotic cells were never observed throughout the entire infection cycle. The HR-like phenotype observed in the ‘UFV 408-28’—M. incognita interaction suggests that the coffee resistance may be mediated by a R-gene based immunity system and may therefore provide new insights for understanding the molecular basis of RKN resistance in perennial crops.     

Pathogenicity and reproductive fitness of <Emphasis Type="Italic">Pratylenchus coffeae</Emphasis> and <Emphasis Type="Italic">Radopholus arabocoffeae</Emphasis> on Arabica coffee seedlings (<Emphasis Type="Italic">Coffea arabica</Emphasis> cv. Catimor) in Vietnam

The pathogenicity and reproductive fitness of Pratylenchus coffeae and Radopholus arabocoffeae from Vietnam on coffee (Coffea arabica) seedlings cv. Catimor were evaluated in greenhouse experiments. The effect of initial population densities (Pi = 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 nematodes per cm³ soil) was studied for both species at different days after inoculation (dai). The data were adjusted to the Seinhorst damage model Y = m + (1-m).z^Pi-T. Tolerance limit (T) for P. coffeae was zero for the height and the diameter of the coffee plants. For the diameter, the T-value for R. arabocoffeae was 25.6 for 30 and 60 dai and 12.8 for 90 and 120 dai. After 4 months T was zero. The low tolerance limits indicate that Arabica coffee is highly intolerant to both nematode species. At the end of the experiment (180 dai), all plants were infected and most were dead when inoculated with R. arabocoffeae at initial densities of 32, 64, 128 and 256 nematodes/cm³ soil. For P. coffeae plant death was already observed at the lowest inoculation densities. Growth of coffee was reduced at all inoculation levels for both species. Pratylenchus coffeae and R. arabocoffeae caused intense darkening of the roots, leaf chlorosis and a strong reduction of root and shoot growth. It was observed that P. coffeae mainly destroyed lateral roots rather than tap roots, whereas R. arabocoffeae reduced tap root length rather than the lateral roots. At the lowest inoculum densities, the reproduction factor of P. coffeae was 2.38 and 2.01 for R. arabocoffeae, indicating that arabica coffee is a host for both species. Plant growth as expressed by shoot height and shoot and root weight measured 60 dai was negatively correlated with nematode (both species) density as expressed by the geometric mean of nematode numbers at 30 and 60 dai.     

Laboratory evaluation of a botanical natural product (<Emphasis Type="Italic">AkseBio2</Emphasis>) against the pear psylla<Emphasis Type="Italic">Cacopsylla pyri</Emphasis>

A botanical natural product,AkseBio2, was evaluated under laboratory conditions for its oviposition deterrent, ovicidal and larvicidal (nymphicidal) effects against the pear psyllaCacopsylla pyri (L.) (Hemiptera: Psyllidae). The product exhibited a strong oviposition deterrent effect for winterform and summerform females and caused a reduction in the total number of eggs laid in both choice and no-choice assays. Significant mortalities in freshly laid eggs (0–48 h) and various nymphal stages of the pest were recorded in toxicity assays. At a concentration of 0.1% (formulation), the highest biological activity of the product was recorded against the young (1st and 2nd) nymphal stages (up to 87.4% mortality) in comparison with the other biological stages of the pest. It was less active against the older (3rd-5th) nymphs, causing 62.1% mortality at the same concentration. In assays with non-target organisms, a significant negative effect was not observed. There were no significant changes on treated plants up to 7 days after treatment in any trial, nor was there any phytotoxicity on plant tissue as a result ofAkseBio2 treatments. The results suggest that the product can be used in psylla control instead of synthetic insecticides and may serve as an integrated pest management (IPM) component in pear orchards. http://www.phytoparasitica.org posting July 14, 2004.     

Host-pathogen interaction of root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> on pepper in the southeast of Spain

The feeder roots of pepper plants (cv. California Wonder) in Campo de Cartagena (southeast Spain) were found to be severely infected by Meloidogyne incognita. Morphometric traits, differential host test and DNA analysis based on PCR were used to characterize the nematode. Naturally and artificially infected pepper plants showed severe yellowing and stunting, with heavily deformed and damaged root systems. Root galls were spherical and commonly contained more than one female and egg masses with eggs. Typical giant cells with a granular cytoplasm and many hypertrophied nuclei were observed in histological preparations. The relationship between initial nematode population density (Pi) and pepper plant growth was tested in greenhouse experiments with inoculum levels that varied from 0 to 64 eggs and second-stage juveniles (J₂) ml⁻¹ soil. A Seinhorst model was fitted to plant height and top fresh weight data of inoculated and non-inoculated plants. The tolerance limit with respect to plant height and fresh top weight of pepper to M. incognita was estimated as 0.85 eggs and J₂ ml⁻¹ soil. The minimum relative values (m) for plant height and top fresh weight were 0.15 and 0.16, respectively, at Pi ≥ 64 eggs and J₂ ml⁻¹ soil. The maximum nematode reproduction rate (Pf/Pi) was 315.4 at an initial population density (Pi) of 4 eggs and J₂ ml⁻¹ soil. The obtained results could be used as a base to establish field experiments that allow strategies to prevent surpassing the threshold of nematodes in fields that are infested.     

Cannibalism of <Emphasis Type="Italic">Brontocoris tabidus</Emphasis> and <Emphasis Type="Italic">Podisus nigrispinus</Emphasis> during periods of pre-release without food or fed with <Emphasis Type="Italic">Eucalyptus cloeziana</Emphasis> plants

Pre-release techniques aim to increase the searching by the predators for their prey. Keeping bug predators for periods without food before their release can increase the search ability of these insects. However, this practice can increase the cannibalism during mass rearing. Brontocoris tabidus (Signoret) and Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) were kept for 12, 24, 36 and 48 h without food or with only Eucalyptus cloeziana plants in order to verify the occurrence of cannibalism on adults and eggs. Insects kept for periods longer than 24 h showed higher cannibalism on adults and eggs and the presence of a E. cloeziana plant decreased the cannibalism during all the treatment periods. These predators preferred to attack first the eggs rather than the adults, probably because of the inability of the former to avoid predation. The pre-release period should not exceed 24 h and plants must be provided to the predators in order to reduce the occurrence of cannibalism and increase the predatory capacity of these natural enemies.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

Reaction of glucosinolate-myrosinase defence system in <Emphasis Type="Italic">Brassica</Emphasis> plants to pathogenicity factor of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The glucosinolate-myrosinase defence system, specific to Brassicales plants, produces toxic volatile compounds during mechanical injury or pathogen attack. The reaction of this system to oxalic acid, known as a pathogenicity factor of Sclerotinia sclerotiorum, is not fully understood. The hydrolysis of glucosinolates was studied at varying conditions in the presence of oxalic acid in the substrate. In a bioassay, colonies of the pathogen were exposed to volatiles from hydrated mustard powder used as a myrosinase and glucosinolate source. The glucosinolate-myrosinase (GSL-M) system was activated in the presence of oxalic acid at a concentration and pH similar to that expected in vivo. Volatile production was inhibited only when the pH fell to 3 or below. It is unlikely that oxalic acid plays a significant role in disarming the GSL-M system during infection of Brassica hosts.     

Inheritance of resistance to Meloidogyne enterolobii in <Emphasis Type="Italic">Psidium guajava</Emphasis> x <Emphasis Type="Italic">P. guineense</Emphasis> hybrid

Inheritance of the resistance to nematodes has been studied on many different crops, however to our knowledge, no data are available for guava species. The basic genetic resistance parameters to Meloidogyne enterolobii are estimated in the current research in order to guide the development of genotypes resistant to the pathogen. The parental plants, F1 and F2 from a Psidium guajava x P. guineense cross were assessed for the presence or absence of galls and for the number of eggs and juveniles in the root system at the 120th and 240th days after inoculation with 10,000 eggs and juveniles of the nematode. At the age of nine years, the P. guineense plant remained without nematode attack symptoms, whereas the maternal plant was destroyed by the pathogen. The F1 generation showed 270 plants with reproduction factor (RF) <0.322, and there were tiny galls in only 16 plants. The segregation for the presence or absence of galls in the root system in generation F2 was 9:7, wherein the χ² values were 0.78 and 2.66, respectively, at the 120th and 240th days after inoculation, whereas the segregation for RF was 15:1, wherein the χ² values were 2.76 and 1.18, respectively, at the 120th and 240th days. These results indicate epistatic interaction between two genes: in RF < 1 only one allele sets the resistance to the pathogen. The broad sense heritability of RF, estimated to the two assessments was 0.97, and it also indicates a simple inheritance of resistance to M. enterolobii.     

Resistance to <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">hirsutum</Emphasis> Dierckx in garlic accessions

Among the factors affecting the quality and yield of garlic production, blue mold caused by -- Penicillium spp. -- is responsible for economical losses in many countries. Allicin, present in garlic bulbs, has been suggested as having antifungal activity against some Penicillium species. This study was conducted to evaluate the response of garlic accessions against Penicillium hirsutum infection and to compare this response with bulb allicin content. Twelve garlic accessions were inoculated with P. hirsutum, and assayed in greenhouse and growth chamber experiments. Plant growth parameters and the fungal production of conidia were evaluated. Significant differences were found among the accessions. Accessions Castaño and Morado were most resistant whereas AR-I-125 and Fuego were always severely affected by the disease. A low correlation was found (r = 0.17) between allicin content and tolerance, indicating that allicin is not the main factor involved in the resistance against P. hirsutum.     

<Emphasis Type="Italic">Erwinia aphidicola</Emphasis> isolated from commercial bean seeds (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

In late 2003, a new disease appeared in protected bean crops in southeastern Spain, causing a decrease of over 50% in production. Several samples of affected plants were collected and analyzed and the agent of this disease was identified as the bacterium Erwinia aphidicola, which had never been described as a pathogen previously. We attempted to determine the possible bacterium transmission through seeds, using 120 commercial bean seeds from the same batch as that used in an affected farm, and 120 seeds from the fruiting plants of the same farm. Seed coats, cotyledons and leaves of plants originating from them, were taken and analyzed. Several of the developed symptoms on plants from commercial and fruiting plant seeds were internervial chlorosis, necrotic pits and rough roots and they coincided with those observed on affected crops. Bacteria present in commercial seed cotyledons were isolated and analyzed by biochemical and molecular tests. Results confirmed the presence of Erwinia aphidicola in four analyzed seeds; moreover, Bacillus simplex/Bacillus muralis, Pseudomonas mendocina, Pseudomonas putida and Paenibacillus polymyxa were also identified.     

Response of tomato rootstocks carrying the <Emphasis Type="Italic">Mi</Emphasis>-resistance gene to populations of <Emphasis Type="Italic">Meloidogyne arenaria,M. incognita and M. javanica</Emphasis>

The response of four Mi-resistance gene tomato rootstocks to seven populations of Meloidogyne was determined in pot tests conducted in a glasshouse. Rootstocks PG76 (Solanum lycopersicum × Solanum sp.) and Brigeor (S. lycopersicum × S. habrochaites) and resistant cv. Monika (S. lycopersicum) were assessed against one population of M. arenaria, three of M. incognita, and three of M. javanica. Rootstocks Beaufort and Maxifort were assessed against one population of M. arenaria, two of M. incognita and two of M. javanica. Rootstock PG76 was highly resistant (reproduction index <10%) to all the populations, whereas rootstock Brigeor and cv. Monika were highly to moderate resistant. Rootstocks Beaufort and Maxifort showed reduced resistance or inability to suppress nematode reproduction, and their responses varied according to the population tested. Beaufort and Maxifort were susceptible to the two populations of M. javanica as Maxifort was to one of M. incognita. The reproduction index of the nematode was higher (P < 0.05) on Maxifort than Beaufort for all root-knot nematode populations.     

Identification of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> and <Emphasis Type="Italic">G. pallida</Emphasis> in the Ukraine by PCR

Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples from the Ukraine. The results show the occurrence of Globodera pallida in the Uzhhorod region (Zakarpatska oblast), where only G. rostochiensis had been previously reported. In the mixed potato cyst nematode (PCN) populations, G. pallida was less prevalent (2–5%) than G. rostochiensis (95–98%). A phylogenetic analysis based on ribosomal DNA internal transcribed spacer sequences showed that the Ukrainian population of G. pallida had >99% sequence identity with other G. pallida pa2/3 isolates from Europe. This study has demonstrated that polymerase chain reaction-mediated amplification of specific regions of the potato cyst nematode genome is not only highly effective as a species diagnostic tool but is also a sensitive method which can be used for taxonomic purposes with cyst collections which vary in age.     

Host-delivered RNAi-mediated root-knot nematode resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis> by targeting <Emphasis Type="Italic">splicing factor</Emphasis> and <Emphasis Type="Italic">integrase</Emphasis> genes

Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.     

<Emphasis Type="Italic">Odontoglossum ringspot virus</Emphasis> causing flower crinkle in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

A new disorder exhibiting flower crinkle on Phalaenopsis orchids bearing white flowers has been observed in Taiwan, China and Japan for several years. This disorder decreased the flower longevity and was considered as a physiological syndrome. The objective of this study was to identify and characterize the real causal agent of this new Phalaenopsis disorder. Five plants of Phalaenopsis hybrids “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) with flower crinkle symptoms were collected and tested by enzyme-linked immunosorbent assay with antisera against 18 viruses. The extract of leaves and flowers from one diseased plant (96-Ph-16) reacted positively only to antiserum against Odontoglossum ringspot virus (ORSV), while those from the other four plants (96-Ph-7, 96-Ph-17, 96-Ph-18 and 96-Ph-19) reacted positively to the antisera against ORSV and Cymbidium mosaic virus (CymMV). Five ORSV isolates, one each from flowers of those five diseased Phalaenopsis orchids, were established in Chenopodium quinoa. A CymMV culture was isolated from the flowers of one of the ORSV/CymMV mix-infected Phalaenopsis orchids (96-Ph-19). To determine the causal agent of the flower crinkle disease, healthy Phalaenopsis seedlings were singly or doubly inoculated with the isolated ORSV and/or CymMV. Results of back inoculation indicated that ORSV is the sole causal agent of the crinkle symptom on petals of Phalaenopsis orchid. The CP gene of the ORSV isolates from this study shared 97.3–100% nucleotide identity and 96.2–100% amino acid identity with those of 41 ORSV isolates available in GenBank. This is the first report demonstrating ORSV as the sole virus causing flower crinkle disease on Phalaenopsis orchids.