Pathogenicity ofPythium species on cucumber in peat-sand,rockwool and hydroponics

Pythium spp. that cause damping-off of seedlings also can cause root rot of older plants and lead to yield reductions. This can be especially severe in soilless cultures where the fungus can spread easily with the nutrient solution. 39Pythium isolates obtained from discolored roots were assayed for their ability to cause damping-off on cucumber seedlings in sand-peat and for their pathogenicity in soilless culture of cucumber in rockwool or hydroponic solution. Isolates ofPythium aphanidermatum, P. irregulare, P. sylvaticum andP. ultimum were highly pathogenic in sand-peat, but onlyP. aphanidermatum strains were pathogenic in soilless conditions and led to root decay, plant death in rockwool culture and growth reduction in hydroponic culture. One strain ofP. aphanidermatum significantly reduced the yield of cucumber grown in rockwool under conditions similar to those of commercial cultures.     

Infection by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> increases production of phenolics in hydroponically grown peppers and predisposes healthy plants to root rot

The type and concentration of phenolics found in the cytosol (free), cell wall (bound), and nutrient solution of hydroponic pepper (Capsicum annuum L.) inoculated with Pythium aphanidermatum (Edson) Fitzp. were compared with non-inoculated controls. The quantities and types of free phenolics found in the roots and nutrient solution were greater in inoculated than in control plants. With regard to bound phenolics, while there was no difference between the types of phenolics in inoculated and control roots, quantities were greater in inoculated roots. High Pressure Liquid Chromatography analyses revealed two compounds in root extracts, and one in nutrient solution extracts that were contributors to root discolouration caused by Pythium aphanidermatum colonization. The compounds were subsequently isolated and identified as 4-hydroxybenzoic acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid) by their High Pressure Liquid Chromatography retention times, UV signature and Nuclear Magnetic Resonance spectroscopic comparisons with pure standards. Addition of various concentrations of 4-hydroxybenzoic acid or vanillic acid to the nutrient solution predisposed healthy plants to infection by zoospores of Pythium aphanidermatum when compared to plants treated with only Pythium aphanidermatum. 4-hydroxybenzoic acid and vanillic acid had little or no effect on colony growth of Pythium aphanidermatum in vitro, but respectively increased and decreased numbers of sporangia produced. These results are the first demonstration of host predisposition to infection by Pythium aphanidermatum caused by phenolic compounds that accumulate in inoculated roots and leach into the nutrient system of hydroponic systems.     

Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield

Pepino mosaic virus (PepMV) was shown to be efficiently transmitted between tomato plants grown in a closed recirculating hydroponic system. PepMV was detected in all plant parts after transmission via contaminated nutrient solution using ELISA, immunocapture RT‐PCR, RT‐PCR, electron microscopy, and by inoculation to indicator plants. Detection of PepMV in nutrient solution was only possible after concentration by ultracentrifugation followed by RT‐PCR. Roots tested positive for PepMV 1–3 weeks after inoculation, and subsequently a rapid spread from the roots into the young leaves and developing fruits was found within 1 week. PepMV was only occasionally detected in the older leaves. None of the infected plants showed any symptoms on fruits, leaves or other organs. Pre‐infection of roots of tomato cv. Hildares with Pythium aphanidermatum significantly delayed PepMV root infections. When mechanically inoculated with PepMV at the 2–4 leaf stage, yield loss was observed in all plants. However, only plants of cv. Castle Rock recorded significant yield losses when infected via contaminated nutrient solution. Yield losses induced by infection with PepMV and/or P. aphanidermatum ranged from 0·4 up to 40% depending on experimental conditions.     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (LAMP) with species-specific primers

A loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA ITS sequence of P. aphanidermatum was developed. Results of a specificity test using 57 strains of Pythium spp. indicated that the LAMP assay gave no cross reactions in other 39 Pythium species, 11 strains of Phytophthora spp. and eight other soil borne pathogens. The detection limit was 10 fg of genomic DNA, which was ten times the sensitivity of the polymerase chain reaction. The LAMP assay was applied to hydroponic solution samples from tomato fields, and the results were compared to those of the conventional plating method. LAMP was observed to be effective for the specific detection of P. aphanidermatum. Furthermore, P. aphanidermatum was detected directly in tomato roots infected with P. aphanidermatum without DNA extraction. The LAMP method established in this study is a simple, sensitive and rapid tool for the detection of P. aphanidermatum.     

Biological control of pythium root rot of chrysanthemum in small-scale hydroponic units

The capacity of several strains of root-colonizing bacteria to suppressPythium aphanidermatum, Pythium dissotocum and root rot was investigated in chrysanthemums grown in single-plant hydroponic units containing an aerated nutrient solution. The strains were applied in the nutrient solution at a final density of 10⁴ CFU ml⁻¹ and 14 days later the root systems were inoculated withPythium by immersion in suspensions of 10⁴ zoospores ml⁻¹ solution. Controls received no bacteria, noPythium, or one of thePythium spp. but no bacteria. Strain effectiveness was estimated based on percent roots colonized byPythium and area under disease progress curves (AUDPC). In plants treated respectively withPseudomonas (Ps.)chlororaphis 63-28 andBacillus cereus HY06 and inoculated withP. aphanidermatum, root colonization by the pathogen was 83% and 72% lower than in the pathogen control, and AUDPC values were reduced by 61% and 65%. ForP. dissotocum, the respective strains reduced root colonization by 87% and 91%, and AUDPC values by 70% and 90%. In plants treated respectively withPs. chlororaphis Tx-1 andComamonas acidovorans C-4-7-28, root colonization byP. aphanidermatum was 84% and 80% lower than in the controls and AUDPC values were reduced by 66% and 57%; these strains did not suppressP. dissotocum. Burkholderia gladioli C-2-74 andC. acidovorans OCR-7-8-38, respectively, suppressed colonization of roots byP. dissotocum by 74% and 86%, and reduced AUDPC values by 60% and 70%, but were ineffective againstP. aphanidermatum. C. acidovorans OCR-7-8-39 reduced colonization and AUDPC values ofP. aphanidermatum by 57% and 42%, respectively.Pseudomonas corrugata 13,Ps. fluorescens 15 and JZ12, and three additional strains ofC. acidovorans were weakly or nonsuppressive againstP. aphanidermatum. Strains that reduced AUDPC values forP. aphanidermatum orP. dissotocum when applied at 10⁴ CFU ml⁻¹ were 11%–39% less effective at 10³ CFU ml⁻¹. Four tested strains (Ps. chlororaphis 63-28,Ps. chlororaphis Tx-1,B. cereus HY06, andB. gladioli C-7-24) in most instances suppressed root colonization and lowered AUDPC values ofP. aphanidermatum when applied at 14, 7 or 0 days before inoculation, but reduction of the respective variables was generally greater when the strains were applied at 14 days (63%–87% and 75%–78%) or 7 days (44%–47% and 31%–88%) than at 0 days (14%–31% and 23%–62%) before inoculation.Ps. chlororaphis Tx-1,Ps. chlororaphis 63-28 andB. cereus HY06 significantly suppressedP. aphanidermatum whether the temperature of the nutrient solution was high (32°C) or moderate (24°C). Taken together, the observations suggest thatPs. chlororaphis 63-28,B. cereus HY06,Ps. chlororaphis Tx-1,B. gladioli C-2-74 andC. acidovorans OCR-7-8-38 have the potential for controlling Pythium root rot in hydroponic chrysanthemums. http://www.phytoparasitica.org posting Jan. 24, 2007.     

Pythium rot of Chinese cabbage (Brassica rapa L. subsp. pekinensis) caused by Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of Chinese cabbage (Brassica rapa L. subsp. pekinensis) in Ibaraki Prefecture every year in early September from 2002 through 2004. The causal fungus was identified as Pythium aphanidermatum (Edson) Fitzpatrick. This is the first report of P. aphanidermatum on Chinese cabbage. A similar disease of Chinese cabbage caused by P. ultimum Trow var. ultimum is known as Pythium rot. We propose adding P. aphanidermatum as a new pathogen of this disease.     

Evaluation of plant growth-promoting rhizobacteria for biological control of pythium root rot of cucumbers grown in rockwool and effects on yield

Three strains ofPseudomonas fluorescens (63-49, 63-28, and 15), one strain ofPseudomonas corrugata (13) and one strain ofSerratia plymuthica (R1GC4) were tested on rockwool-grown cucumbers for their ability to reduce Pythium root-rot caused byPythium aphanidermatum. These strains were previously selected for biocontrol ability from collections of >4000 bacteria. Strains 63-49 and 63-28 were tested on cucumber plants grown in rockwool in two replicatedPythium-inoculated trials conducted in British Columbia (B.C). Another inoculated, replicated trial was conducted in Quebec with all five strains. Cucumber yields (fruit number and weight) were measured over a ten-week harvest period. Strain 63-49 caused an early promotion of plant growth and increased cucumber yields at early harvests. No measurable effect ofPythium inoculation on disease development was observed in the Quebec trial, due to unfavourable cool weather. However, 63-49 significantly increased the total number of cucumbers (12%) and cucumber weight (18%), compared to the non-treated control. Strains 13, 15 and R1GC4 slightly increased the cumulative cucumber yields, but strain 63-28 had no effect. In the B.C. trial, inoculation withP. aphanidermatum reduced the number and weight of cucumbers by 27%. Treatments ofPythium-inoculated cucumbers with 63-49 significantly increased fruit number and weight by 18%, compared to thePythium-inoculated control. Strain 63-28 increased the cumulative number of cucumbers over time, compared to thePythium-inoculated control, but the increase was less than with 63-49. The use ofPseudomonas spp. in rockwool-grown cucumbers can increase yields, both in the presence and absence of Pythium root rot, and with variable seasonal conditions and disease pressures.     

The development of a PCR-based method for detecting <Emphasis Type="Italic">Puccinia striiformis</Emphasis> latent infections in wheat leaves

Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici is one of the most important diseases on wheat worldwide, especially in temperate regions with cool moist weather conditions. A rapid and reliable detection of the pathogen in latent infected wheat leaves during overwintering of the fungus in the dormant stage will contribute to determine the initial inoculum potential and thus to predict early outbreak and to improve effective management of the disease. To achieve this aim, a PCR-based method was developed for specific and sensitive detection of P. striiformis. Specific primers were designed according to a genome-specific sequence of P. striiformis. To evaluate the specificity of the primers, seven different isolates and races of P. striiformis as well as six other pathogens of wheat were tested. All isolates of P. striiformis yielded a distinct band of a fragment of 470 bp, while using DNA of the other wheat pathogens as a template no amplification product was detected. The sensitivity of the primers was tested using serial dilutions of total DNA from P. striiformis; the limit of detection was 10 pg of DNA. Using extracts from P. striiformis-infected wheat leaves, the fungus could be determined in the leaves before symptoms appeared. The stripe rust could also be detected in the dormant stage by the PCR assay in samples of wheat leaves taken during the winter season. The application of the PCR assay may be useful for rapid and reliable detection of P. striiformis in latent infected leaves of overwintering wheat plants.     

Effects of inoculum density,plant age and temperature on disease severity caused by pythiaceous fungi on several plants

Alfalfa, maize, sorghum and sugarbeet plants were inoculated with zoospores ofPhytophthora andPythium species in order to assess the effects of inoculum density, plant age and temperature on disease severity. Seedlings were grown axenically in test tubes and inoculated with zoospore suspensions. Disease severity was assessed by measuring the root growth and discoloration of treated and control seedlings. The incremental root length of all plants decreased and root discoloration increased as inoculum concentration of the pathogen increased. Changes were more intensive among low levels of zoospore concentrations and no significant differences in disease severity were found for inoculum densities higher than 10⁴ zoospores ml^-1. Disease severity was negatively related to plant age. Disease development on sugarbeet seedlings infected withPythium andPhytophthora species was affected by temperature, but the pattern of response was determined by the pathogen’s temperature preferences. The incremental root length decreased as temperature increased up to 25°C. The effect ofPythium dissimile andPhytophthora cactorum on root length was significantly lower at 35°C than at 25°C, whereasPythium aphanidermatum andPhytophthora nicotianae caused significant damage to roots even at 35°C. http://www.phytoparasitica.org posting Dec. 3, 2001.     

Cloning of a Genomic DNA Encoding Caffeoyl-coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase of Citrus

In recent studies, we found that Apll (a PthA homologue) bound to three citrus proteins. Amino acid sequence analysis revealed that one of the target proteins was homologous to that of S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT), an enzyme which is specific for the substrate trans-caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA. From the consensus nucleotide sequences of CCoAMT genes, primers were chosen for PCR amplification of this gene from citrus total DNA. Two selected DNA fragments of 1.0 kb and 2.0 kb were obtained. These fragments were used as the probe to screen a citrus library. One clone, pCCl00, contained a 1.0-kb SalI fragment that hybridized to the probes. The nucleotide sequence of this fragment was determined in both directions. In this fragment, there was an open reading frame of 232 amino acids interrupted by an intron of 106 nt, and the deduced amino acid sequence had 95.9% homology to tobacco CCoAMT. Southern blot analysis of total citrus DNA showed that four EcoRI fragments hybridized to the probes, suggesting the presence of more than one copy of CCoAMT in the citrus DNA. Received 4 November 1999/ Accepted in revised form 26 November 1999     

Pythium rot of figmarigold (<Emphasis Type="Italic">Lampranthus spectabile</Emphasis>) caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

A new leaf rot disease was found on the leaves of figmarigold (Lampranthus spectabile). The causal organism, identified as Pythium aphanidermatum was found to cause the same symptoms after artificial inoculation and was then reisolated from the inoculated plants. We propose to name the disease Pythium rot of figmarigold.     

Enhancing Resistance of Tomato and Hot Pepper to Pythium Diseases by Seed Treatment with Fluorescent Pseudomonads

Twenty isolates of fluorescent pseudomonads were evaluated for their ability to control damping-off in tomato (Lycopersicon esculentum) and hot pepper (Capsicum annuum). These isolates were characterized as Pseudomonas fluorescens and Pseudomonas putida. Two isolates, PFATR and KKM 1 belonged to P. putida and the remaining 18 isolates belonged to P. fluorescens. Among these isolates, P. fluorescens isolate Pf1 showed the maximum inhibition of mycelial growth of Pythium aphanidermatum and increased plant growth promotion in tomato and hot pepper. P. fluorescens isolate Pf1 was effective in reducing the damping-off incidence in tomato and hot pepper in greenhouse and field conditions. Isolate Pf1 was further tested for its ability to induce production of defense-related enzymes and chemicals in plants. Earlier and increased activities of phenylalanine ammonia lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) were observed in P. fluorescens Pf1 pretreated tomato and hot pepper plants challenged with Pythium aphanidermatum. Moreover, higher accumulation of phenolics was noticed in plants pretreated with P. fluorescens isolate Pf1 challenged with Pythium aphanidermatum. Thus, the present study shows that in addition to direct antagonism and plant growth-promotion, induction of defense-related enzymes involved in the phenyl propanoid pathway collectively contributed to enhance resistance against invasion of Pythium in tomato and hot pepper.     

Monitoring by real-time PCR of three water-borne zoosporic Pythium species in potted flower and tomato greenhouses under hydroponic culture systems

The high-temperature-tolerant Pythium species P. aphanidermatum, P. helicoides, and P. myriotylum cause serious diseases in many crops under hydroponic culture systems in Japan. Control of the diseases is difficult because these zoosporic pathogens spread quickly. In this study, a real-time PCR method was developed for monitoring the spread of zoospores of the three pathogens. Specific primers and TaqMan probes were established using the internal transcribed spacer regions of the rDNA. Specificity was confirmed using known isolates of each species and closely related non-target species. The sensitivity of DNA detection was 10 f. for each pathogen. 10 f. DNA corresponded to 4 P. aphanidermatum, 3 P. myriotylum, and 4 P. helicoides zoospores, respectively. Therefore, this real-time PCR method was used to evaluate and monitor zoospores in the nutrient solutions of ebb-and-flow irrigation systems for potted flower production and closed hydroponic culture systems for tomato production. The results indicated that the pathogens were present in the hydroponic culture systems throughout the year, and spread before disease occurrence.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Cottony leak of scarlet runner bean caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

Severe cottony leak was found on stems of scarlet runner bean (Phaseolus coccineus) in Ibaraki Prefecture in August 2006. The causal fungus was identified as Pythium aphanidermatum. The name cottony leak of scarlet runner bean (Watagusare-byo in Japanese) is proposed for this new disease.     

Biocontrol traits of plant growth suppressive arbuscular mycorrhizal fungi against root rot in tomato caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

Arbuscular mycorrhizal (AM) fungi known to cause plant growth depressions in tomato were examined for their biocontrol effects against root rot caused by Pythium aphanidermatum. The main hypothesis was that plant growth suppressive AM fungi would elicit a defence response in the host plant reducing Pythium root rot development. To test this hypothesis a fully factorial experiment was performed with AM fungi (Glomus intraradices, G. mosseae, G. claroideum or nonmycorrhizal), Pythium (± P. aphanidermatum) and harvest (7 and 14 days after pathogen inoculation (dapi)) as the main factors. Two weeks after AM fungi inoculation, roots were challenged with P. aphanidermatum. Variables evaluated at each harvest were root colonization levels of the interacting fungi, plant growth responses, and expression of a plant pathogenesis related protein gene (PR-1). All of the tested AM fungi caused marked growth suppressions, but did not affect PR-1 gene expression or the phosphorous concentration in the host plant. Plants singly inoculated with P. aphanidermatum had an increased PR-1 expression and phosphorous concentration. Among the AM fungi included in the study only G. intraradices reduced the pathogen root infection level, measured both in terms of Pythium ELISA and by recovery on selective media and only at the first harvest. Likewise, P. aphanidermatum root infection reduced colonization levels of G. intraradices, but not that of the two other AM fungi. In conclusion, plant growth suppressive AM fungi may offer plant beneficial traits in terms of biocontrol of root cortical pathogens.     

Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum

Root and crown rot of cucumber caused by Pythium aphanidermatum can be suppressed by various rhizobacteria or PGPR (plant growth-promoting rhizobacteria). When cucumber roots were treated with Pseudomonas corrugata 13 or Pseudomonas aureofaciens 63–28, phenylalanine ammonia-lyase (PAL) activity was stimulated in root tissues in 2 days and this activated accumulation lasted for 16 days after bacterization. Peroxidase (PO) and polyphenol oxidase (PPO) activities were increased in roots 2–5 days after bacterization with P. corrugata strain 13. After bacterized cucumber roots were challenged with P. aphanidermatum, the enzyme activities of PAL, PO and PPO increased as the disease developed on the roots. These accumulations peaked 4–6 days after pathogen inoculation. A split root system demonstrated that the three enzymes could be systemically induced by the Pseudomonas strains 63–28 and 13, as well as P. aphanidermatum. Furthermore, isoperoxidase native PAGE (polyacrylamide gel electrophoresis) analysis indicated that the peroxidase isomer forms in cucumber roots induced by rhizobacteria were different from that in roots infected with P. aphanidermatum. These results suggest that the plant defense enzymes could be stimulated in cucumber roots which have been colonized by non-pathogenic rhizobacteria or in a compatible interaction between cucumber and P. aphanidermatum. The mechanisms of PO activation by the rhizobacteria may be different from those of pathogen infection.     

Pythium rot of chingensai ( Brassica campestris L. chinensis group) caused by Pythium ultimum var. ultimum and Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of chingensai (Brassica campestris L. chinensis group) in Okayama Prefecture in 2000. The causal fungi were morphologically identified as Pythium ultimum Trow var. ultimum and P. aphanidermatum (Edson) Fitzpatrick. This is the first report of rot caused by Pythium species on chingensai. We named this disease Pythium rot of chingensai.     

Pcr-amplification of tomato yellow leaf curl virus (TYLCV) DNA from squashes of plants and whitefly vectors: Application to the study of TYLCV acquisition and transmission

DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was amplified from squashes of infected tomato plants and of viruliferous vectors using the polymerase chain reaction (PCR). Samples of infected tissues as small as 1 mm² were squashed onto a nylon membrane. A 1 × 2 mm strip containing the squash was introduced into a 25 µl PCR reaction mix. The reaction products were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from individual viruliferous whiteflies. The same squash could be used several times to amplify different virus DNA fragments with various sets of primers. Thus plant and insect squashes can be used as templates for the amplification of geminiviral DNA with no need to prepare tissue extracts or purify nucleic acids. The squash-PCR procedure was applied to study whitefly transmission of TYLCV. Tomato plants were inoculated by placing a single viruliferous insect in the center of a young leaflet. In some plants TYLCV DNA was detected at the site of inoculation as early as 5 min after the beginning of the access feeding and in all plants after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the access feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min.