Detection by PCR of Candidatus Liberibacter asiaticus, the bacterium causing citrus huanglongbing in vector psyllids: application to the study of vector–pathogen relationships

Citrus huanglongbing (HLB), previously called greening, is a serious citrus disease in Asia, eastern and southern Africa. It is caused by Candidatus Liberibacter asiaticus (Las), a phloem-limited, nonculturable bacterium transmitted by the Asian citrus psyllid ( Diaphorina citri ) in Asia. A PCR-based assay was developed for monitoring Las in vector psyllids using a rapid DNA extraction from psyllid bodies and PCR amplification. The entire procedure for Las detection in psyllids can be completed within 5 h. Using this method, Las can be accurately detected in psyllid adults as well as nymphs in different instar stages. The assay is sensitive enough for Las detection in single-psyllid extract from adult, fifth, fourth and third instars. In a transovarial transmission experiment, Las was not detected in eggs or in offspring produced by Las-carrying psyllid females. In a retention test, the Las-carrying psyllids remained Las-positive for 12 weeks after they were moved to common jasmine orange, a Las-immune plant. From these experimental results it was concluded that Las persists in the Asian citrus psyllid vector, but is not transovarially transmitted by the vector. These data help in understanding epidemiological characteristics of Las and psyllids in citrus HLB.     

柑橘黄龙病病原研究进展

本文对柑橘黄龙病(HLB)的发现、认识过程及病原菌的分类、检测鉴定技术进行了综述。HLB是一种十分严重的柑橘病害,主要通过木虱和嫁接进行传播,在不同的地区和国家具有不同的名字,已统一命名为HLB。对引起HLB的原因探索经历了一个漫长的过程,从水害、缺素、镰刀菌、病毒到类菌原体,最后确定为细菌。该病原属于α-亚纲,韧皮部杆菌属,分为亚洲种、非洲种和美洲种3个种,可以通过PCR技术进行检测鉴定。同时,对韧皮杆菌的人工培养以及与黄龙病相关植原体的最新研究进展进行了阐述。     

A graft-based chemotherapy method for screening effective molecules and rescuing huanglongbing-affected citrus plants

Huanglongbing (HLB) is the most devastating disease of citrus. The global citrus industry is in urgent need of effective chemical treatments for HLB control because of its rapid spreading worldwide. Due to the fastidious nature of the pathogens, and the poor permissibility of citrus leaf surfaces, effective screening of chemicals for the HLB control can be challenging. In this study, we developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the best HLB-affected scion-rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared with grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. These findings are useful not only for chemical treatments but also for graft-based transmission studies in HLB and other Liberibacter diseases.     

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts,rootstocks and the Asian citrus psyllid

The use of proper management strategies for citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR. Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (10⁴–10⁷ copy numbers μL^?1) and Las‐carrying percentages (LCP; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.     

柑桔木虱传递柑桔黄龙病的初步研究

柑桔木虱是柑桔黄龙病主要媒介昆虫。几年来的试验初步表明:无病植株上繁殖木虱成虫,在病树上吸毒20—30天以上就可传病。黄龙病病原在成虫体内约经20—30天循回期、带毒后木虱成虫,虫口密度大(20头/苗以上),吸食传毒1天即可发病;虫口密度小(1—5头/苗),吸食传毒7—14天也能传病。潜伏期一般2—8个月,个别株2年才发病,病梢于冬季回接,3—5个月后也表现出和虫传发病苗相同的斑驳状病症。     

<Emphasis Type="Italic">In planta</Emphasis> multiplication and graft transmission of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus’ revealed by Real-Time PCR

The bacterium Candidatus Liberibacter asiaticus (CLas) is associated with huanglongbing (HLB) in citrus in many countries. Despite the fact that many characteristics of the disease are known, the rate of multiplication of the bacterium within an infected tree is still poorly understood. To study this feature, we used the quantitative Real-Time polymerase chain reaction (Q-PCR) assay to follow and to quantify the multiplication of CLas in grafted infected young sweet orange plants. The rate of infection by grafting reached 100% at 120 days post-inoculation (dpi) showing that grafting could easily transmit CLas. A well-adjusted linear regression equation describing the bacterial growth in planta was obtained independently with measurements taken using repeated sampling in the same plant or different plants through the analysed period. The bacterial population, measured as copy number (CN) of the 16S rDNA target gene g⁻¹ of tissue, increased 10,000 times from 10³ at 30 dpi to approximately 10⁸ CN at 240 dpi indicating that CLas multiplication was fastest in young citrus plants. We observed a direct relationship between the concentration of pathogen and the expression of symptoms. Yellowed leaves or shoots, are commonly the first observed symptom of HLB, and were present in trees with a low amount of bacteria (10⁵ CN g⁻¹). Blotchy mottle symptoms were observed in trees with 10⁷ CN g⁻¹ of bacteria after 180 dpi. Buds taken from infected, but non-symptomatic branches were grafted on Rangpur lime and resulted in transmission rates ranging from 10 to 60%.     

Incidence of ‘Candidatus Liberibacter europaeus’ and phytoplasmas in Cacopsylla species (Hemiptera: Psyllidae) and their host/shelter plants

Psyllids, as vectors of phloem-restricted plant pathogens, are serious agricultural pests. Fruit tree phytoplasmas are transmitted by different Cacopsylla spp., while other psyllids are known vectors of liberibacters. Recently, the bacterium ??Candidatus Liberibacter europaeus?? was found in pear trees and in Cacopsylla pyri (Linnaeus), the vector of ??Ca. Phytoplasma pyri??. This new species does not cause symptoms in plants and is probably a symbiont rather than a pathogen. Based on these findings and the assumption that ??Ca. Liberibacter europaeus?? is widespread, we studied its distribution in the genus Cacopsylla and in the respective host and shelter plants (where psyllids aestivate and overwinter), as well as its possible co-presence with ??Ca. Phytoplasma?? spp. We tested 14 Cacopsylla species and 11 plant species from northwestern Italy, Hungary and Israel, characterized by warm oceanic, temperate continental and warm Mediterranean climatic conditions, respectively. ??Ca. Liberibacter europaeus?? was common within the Cacopsylla genus, being present in nine of the 14 species screened as well as in most host plants, whereas none of the shelter plants tested positive for this bacterium. Altogether, these findings indicate the presence of ??Ca. Liberibacter europaeus?? in continental zones, whereas it does not seem to be widespread in the Mediterranean region. Furthermore, lack of specific symptoms in all infected plants confirms an endophytic relationship with this bacterium, while its abundance in insects suggests a beneficial role for the host. Co-infections with phytoplasmas, observed in insects and plants, require further study to evaluate the possible interactions between them.     

In planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain reaction (PCR) and real-time PCR

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.     

Detection of 'Candidatus Liberibacter asiaticus' in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of 'Ca. L. asiaticus' in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of 'Ca. L. asiaticus' in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.     

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.     

Interaction between Likubin Bacterium and Phytophthora parasitica in Citrus Hosts

Infection with Likubin bacterium (LB) followed by Phytophthora parasitica increased the mortality of sour orange and pummelo seedlings, and enhanced the P. parasitica-induced root rot in all the four types of citrus tested. The LB-induced enhancement of root infection by P. parasitica was apparent within 1h of exposure to zoospore suspension. The enhancement of P. parasitica-induced root rot was affected by the infection sequence. Inoculation of sour orange seedlings with LB before P. parasitica was more effective in increasing P. parasitica-induced root rot than LB and P. parasitica concomitantly or LB after P. parasitica. Grafting P. parasitica susceptible scions of ponkan (Citrus reticulata) onto P. parasitica-tolerant rootstocks of sour orange greatly increased the susceptibility of rootstocks to P. parasitica. Results also demonstrate the enhancement of LB-induced symptoms by P. parasitica in citrus plants.     

Chemical compounds effective against the citrus Huanglongbing bacterium 'Candidatus Liberibacter asiaticus' in planta

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide and is threatening the survival of the Floridian citrus industry. Currently, there is no established cure for this century-old and emerging disease. As a possible control strategy for citrus HLB, therapeutic compounds were screened using a propagation test system with 'Candidatus Liberibacter asiaticus'-infected periwinkle and citrus plants. The results demonstrated that the combination of penicillin and streptomycin (PS) was effective in eliminating or suppressing the 'Ca. L. asiaticus' bacterium and provided a therapeutically effective level of control for a much longer period of time than when administering either antibiotic separately. When treated with the PS, 'Ca. L. asiaticus'-infected periwinkle cuttings achieved 70% of regeneration rates versus <50% by other treatments. The 'Ca. L. asiaticus' bacterial titers in the infected periwinkle plants, as measured by quantitative real-time polymerase chain reaction, decreased significantly following root soaking or foliar spraying with PS. Application of the PS via trunk injection or root soaking also eliminated or suppressed the 'Ca. L. asiaticus' bacterium in the HLB-affected citrus plants. This may provide a useful tool for the management of citrus HLB and other Liberibacter-associated diseases.     

Effect of different host plants on the development time and parasitization rate of eretmocerus dehachi rose and rosen (hymenoptera: aphelinidae) withparabemisia myricae (Kuwana) (homoptera: aleyrodidae) as insect host

The effect of six citrus and five non-citrus host plants on the developmental time ofEretmocerus dehachi Rose and Rosen (Hymenoptera: Aphelinidae), the parasitoid ofParabemisia myricae (Kuwana) (Homoptera: Aleyrodidae), was studied under laboratory conditions. On citrus host plants, the mean developmental time from egg to adult was shortest on lemon with 14.6 days and longest on trifoliate orange with 26.3 days. On non-citrus host plants the developmental time ranged between 16.3 days on vine and 23.0 days on pomegranate. Among the various host plants tested,E. dehachi preferredP. myricae on sweet orange. A high mortality ofP. myricae due to host feeding and parasitization was observed on sweet orange and grapefruit among citrus host plants and on rose among non-citrus host plants.     

Effect of different host plants on the development time and parasitization rate of eretmocerus dehachi rose and rosen (hymenoptera: aphelinidae) withparabemisia myricae (Kuwana) (homoptera: aleyrodidae) as insect host

The effect of six citrus and five non-citrus host plants on the developmental time ofEretmocerus dehachi Rose and Rosen (Hymenoptera: Aphelinidae), the parasitoid ofParabemisia myricae (Kuwana) (Homoptera: Aleyrodidae), was studied under laboratory conditions. On citrus host plants, the mean developmental time from egg to adult was shortest on lemon with 14.6 days and longest on trifoliate orange with 26.3 days. On non-citrus host plants the developmental time ranged between 16.3 days on vine and 23.0 days on pomegranate. Among the various host plants tested,E. dehachi preferredP. myricae on sweet orange. A high mortality ofP. myricae due to host feeding and parasitization was observed on sweet orange and grapefruit among citrus host plants and on rose among non-citrus host plants.     

Effects of huanglongbing on fruit quality of sweet orange cultivars in Brazil

Citrus huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus and Ca. L. americanus and transmitted by the Asian psyllid Diaphorina citri, is the most serious disease of citrus worldwide because of crop devastation and difficulty to control. Since 2004, approximately 3 million trees were eliminated in attempts to limit its spread in Brazil. Where HLB becomes endemic, the disease progression in the orchard and the increasing symptom severity throughout the tree canopy can be relatively fast, greatly reducing the economic life of affected orchards because of tree decline and yield reduction. Although the majority of the fruit from symptomatic branches drop before harvest, a significant amount of affected fruit remain attached, are available for harvest, and can affect juice quality. To quantify and compare the effects of HLB on fruit quality of the most important sweet orange cultivars grown in São Paulo State, 4–6 year-old sweet orange trees from 26 blocks (two of ‘Valencia Americana’, eight of ‘Hamlin’, four of ‘Westin’, seven of ‘Pera’, and five of ‘Valencia’) were selected prior to harvest. In each block, 14–21 HLB-symptomatic trees were chosen. In each tree, the quality of 20 fruit normal in appearance from asymptomatic branches and 20 symptomatic fruit from symptomatic branches were assessed. In general, compared to normal fruit, the symptomatic fruit were small, light, more acidic, and had lower juice percentage, Brix, total soluble solids per box, total soluble solids per fruit, and Brix/acidity ratio. These effects of fruit quality were less pronounced on early and mid season sweet orange cultivars than on late season cv. Valencia.     

中国不同地区亚洲韧皮杆菌遗传多样性分析

 柑桔黄龙病是最严重的柑桔病害之一,现已威胁世界柑桔产业。已被证实在中国南部该病害通过嫁接和木虱传播。因此黄龙病的流行病学研究就显得尤为重要。本研究中,基于外膜蛋白(omp)基因的PCR-RFLP的方法被用来研究中国南部各柑桔主产区的亚洲韧皮部杆菌的遗传多样性。研究采用不同的限制性内切酶消化各地区的亚洲韧皮部杆菌的omp基因产生了不同的RFLP指纹图谱,并对各地亚洲韧皮部杆菌的omp基因进行克隆测序然后构建系统发育树。结果显示:中国不同地理区域的亚洲韧皮部杆菌具有一定的遗传多样性,即便在某一特定的地区也有一定的差异。序列分析显示中国地区的亚洲韧皮部杆菌有很高的同源性。这些结果对揭示中国柑桔黄龙病的流行病学及制定科学有效的防治策略具有一定的指导意义。     

Differential colonization patterns of Xylella fastidiosa infecting citrus genotypes

Xylella fastidiosa is a phytopathogenic bacterium that causes disease in many different crops worldwide. In Brazil, X. fastidiosa subsp. pauca causes citrus variegated chlorosis (CVC), which is a disease responsible for economic losses in the citrus agribusiness. Variable host responses to bacterial colonization and disease development have been observed. This work studies the colonization processes of a pathogenic GFP‐labelled X. fastidiosa citrus strain in sweet orange (susceptible) and tangor (resistant) parents and two resulting hybrids that exhibited contrasting responses to CVC. Xylella fastidiosa showed increased populations and movement in the susceptible genotypes, but slower compared to other hosts such as grapevine. Scanning electron microscopy revealed that the predominant pitted stem morphology in citrus makes the bacterial movement difficult. In susceptible genotypes X. fastidiosa can move from the primary to the secondary xylem, whilst it is confined to the primary xylem in resistant plants. Associated with this is an induction of lignification that occurs earlier in the resistant genotypes when in the presence of the pathogen, and represents a genetic mechanism that leads to formation of a physical barrier, impairing bacterial colonization.     

Liberibacters associated with orange jasmine in Brazil: incidence in urban areas and relatedness to citrus liberibacters

Two surveys (2005/2006 and 2009) were conducted in the state of São Paulo, Brazil, to investigate the incidence of ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’, two liberibacters associated with citrus huanglongbing (HLB) disease and both transmitted by Diaphorina citri, in orange jasmine (Murraya exotica), a widespread ornamental tree in cities and villages. The graft‐transmissibility of the two species, and their DNA relatedness to citrus‐associated liberibacters, were also investigated. Quantitative PCR was applied to PCR‐positive orange jasmine and HLB‐positive citrus growing in backyards and orchards to assess their inoculum source potentials. Liberibacters were detected in 91 of 786 sampled orange jasmine plants in 10 of 76 sampled locations. PCR‐positive trees exhibited yellow shoots and/or dieback symptoms indistinguishable from those on PCR‐negative trees. ‘Candidatus Liberibacter americanus’ was more common in 2005/2006 (96·6%) and ‘Ca. L. asiaticus’ in 2009 (84·8%). rplJ nucleotide sequences were identical within all populations of either species. Graft transmission succeeded only in homologous host combinations, including ‘Ca. L. americanus’ (2/10) from/to orange jasmine and ‘Ca. L. americanus’ (5/18) and ‘Ca. L. asiaticus’ (5/9) from/to citrus. Symptoms were mild and developed less rapidly in orange jasmine than in citrus, probably as a result of lower liberibacter multiplication rates. Respective titres of ‘Ca. L. americanus’ and ‘Ca. L. asiaticus’ in orange jasmine averaged 4·3 and 3·0 log cells g^?1 tissue, compared with 5·5 and 7·3 in citrus. The results indicate that orange jasmine does not favour liberibacter multiplication as much as citrus. However, its importance in HLB epidemics should not be underestimated as it is a preferred host of D. citri and is not under any strict tree‐eradication programme or measures for insect control.     

乙氧氟草醚对脐橙的杀梢效果及对柑橘木虱栖息分布的影响

柑橘木虱偏好产卵于柑橘嫩梢新芽上,在统一抹芽放梢后及时施药防治木虱,成为防控柑橘黄龙病的重要措施。近年通过杀梢剂处理杀灭新发嫩梢代替人工抹梢受到果农的关注和使用,并已成为防治黄龙病的重要技术手段。本研究以纽荷尔脐橙为供试树种,喷施不同质量浓度的乙氧氟草醚药液,探究其杀梢效果及对柑橘木虱栖息分布的影响,并建立了嫩梢萎蔫的分级标准和木虱栖息分布的研究方法。结果表明:乙氧氟草醚对脐橙树苗和定植树有相似的杀梢效果,质量浓度为10 mg/L时即有一定的杀梢作用,40~55 mg/L时杀梢效果最佳,速效性优异,对柑橘叶片和成熟期果实无药害。采用40和55 mg/L的乙氧氟草醚药液对树苗喷雾,3 d后,嫩梢长度抑制率分别为86.1%和124.7%,嫩梢直径抑制率分别为83.8%和94.8%;7 d后,校正杀梢率分别为93.7%和84.1%,木虱校正死亡率分别为7.8%和21.4%,木虱栖息抑制率分别为81.0%和84.4%。采用40、55 mg/L的乙氧氟草醚药液对定植树喷雾,3 d后嫩梢长度抑制率分别为85.9%和118.8%,嫩梢直径抑制率分别为83.9%和104.1%;7 d后校正杀梢率分别为96.6%和82.4%。嫩梢萎蔫可使嫩梢上的木虱虫卵和若虫死亡,对成虫死亡率影响小,但栖息环境发生迁移。因此,乙氧氟草醚施用于脐橙可快速杀梢,减少木虱的食物来源,有效控制木虱种群数量。     

Virus transmission by host-specific strains ofOlpidium bornovanus andOlpidium brassicae

Zoospores of 12 isolatesO. bornovanus from geographically diverse sites and representing the three host specific cucurbit strains were tested as vectors for seven viruses using watermelon bait plants and the in vitro acquisition method. All isolates of the cucumber, melon, and squash strains transmitted melon necrotic spot carmovirus (MNSV) and cucumber necrosis tombusvirus (CNV) but none transmitted petunia asteroid mosaic tombusvirus (PAMV) or tobacco necrosis necrovirus (TNV). The isolates varied as vectors of three other carmoviruses: cucumber leaf spot virus (CLSV); cucumber soil borne virus (CSBV); and squash necrosis virus (SqNV). All cucumber isolates transmitted CLSV and SqNV but not CSBV. Some of the melon isolates transmitted CLSV and SqNV but none transmitted CSBV. Two squash isolates transmitted CSBV and SqNV but not CLSV. Two isolates ofO. brassicae transmitted only TNV and a third did not transmit any of the viruses. The species of bait plant sometimes affected transmission. The most efficient vector strains ofO. bornovanus, as determined by reducing zoospores and virus in the inoculum, were the cucumber strain for CLSV; the cucumber strain for CNV if cucumber was the bait plant or melon strain if watermelon was the bait plant; and the squash strain for SqNV. The plurivorous strain ofO. brassicae was the most efficient vector of TNV.Olpidium bornovanus is the first vector reported for CSBV and is confirmed as a vector of SqNV. It is proposed that all carmoviruses may have fungal vectors.Ligniera sp. did not transmit any of the viruses in one attempt.Abbreviations CLSV cucumber leaf spot virus - CNV cucumber necrosis virus - CSBV cucumber soil borne virus - MNSV melon necrotic spot virus - PAMV petunia asteroid mosaic virus - SqNV squash necrosis virus - TNV tobacco necrosis virus - TBSV tomato bushy stunt virus