Different symptoms in carrots caused by male and female carrot psyllid feeding and infection by ‘Candidatus Liberibacter solanacearum’

Carrot psyllid Trioza apicalis was recently found to carry the plant pathogenic bacterium ‘Candidatus Liberibacter solanacearum’ (CLs). To confirm the transmission of bacteria by the psyllids and to dissect the symptoms caused in carrot plants by psyllid feeding and CLs infection, a greenhouse experiment with single psyllids feeding on separate plants was performed. A positive correlation was found between the amount of CLs bacteria in the psyllids and in the corresponding plants exposed to feeding, indicating CLs transmission. The female psyllid feeding caused more severe damage than male feeding, and resulted in a substantial decrease in the root weight. Female psyllid feeding also significantly reduced the carrot leaf weight and increased the number of curled leaves. The number of curled leaves was also increased by the nymphs when their number exceeded 10 per plant. A high titre of CLs bacteria significantly reduced root weight, while not affecting the weight or number of the leaves. However, the amount of CLs correlated with the number of leaves showing discolouration symptoms. Microscopy of infected carrot plants revealed that the phloem tubes throughout the whole plant, from leaf veins to the root tip, were colonized by bacteria. The bacterial cells appeared to be long and thin flexible rods with tapering ends and a transversally undulated surface. Microscopy also revealed collapsed phloem cells in the infected carrots. Damage in the phloem vessels is likely to reduce the sucrose transport from source leaves to the root, explaining the observed leaf discolouration and reduction in root weight.     

A new haplotype of “Candidatus Liberibacter solanacearum” identified in the Mediterranean region

“Candidatus Liberibacter solanacearum”, a phloem-limited and Gram-negative bacterium that is spread from infected to healthy plants by psyllid insect vectors, is an economically important pathogen of solanaceous and carrot crops in the Americas, New Zealand and Europe. Three haplotypes of “Ca. L. solanacearum” have previously been described, two (LsoA and LsoB) in relation to solanaceous crops in the Americas and New Zealand and the third (LsoC) to carrots in Finland. Herein, we describe a fourth haplotype of this ‘Candidatus Liberibacter’ species (LsoD), also associated with carrots, but from Spain and the Canary Islands and vectored by the psyllid Bactericera trigonica. In addition, LsoC was confirmed in carrot and psyllid samples recently collected from Sweden and Norway. Phylogenetic analysis of the 16S rRNA gene suggests that two of the haplotypes, one in the Americas and the other in northern Europe are closer to each other in spite of a large geographic separation and host differences. Furthermore, during this study, potatoes with symptoms of zebra chip disease recently observed in potato crops in Idaho, Oregon and Washington states were analyzed for haplotype and were found to be positive for LsoA. This liberibacter haplotype was found in psyllids associated with the diseased potato crops as well. This finding contrasts with an earlier report of LsoB from psyllids in Washington which came from a laboratory colony originally collected in Texas.     

PCR‐based identification of Pythium spp. causing cavity spot in carrots and sensitive detection in soil samples

On the basis of ITS sequences PCR primers were designed for the identification of the five Pythium species found to be most important for the development of carrot cavity spot in Norway: P. intermedium, P. sulcatum, P. sylvaticum, P. violae and P. ‘vipa’. The P. ‘vipa’ isolates had a unique ITS sequence, differed morphologically from all other Pythium isolates, and thus probably represent a new species. The PCR primers were species‐specific with no cross‐reaction to other Pythium species or to fungal isolates from carrot tested. The detection limits varied for the different primer pairs. The two most sensitive assays allowed detection of as little as 5 fg DNA. All five Pythium species could be detected in lesions from diseased carrots. Weak positive signals were obtained from some carrot samples without symptoms. PCR assays allowed detection of pathogens in soil. In samples of soil known to produce cavity spots on cropped carrots, strong signals were obtained. In several soil samples more than one of the five Pythium species could be detected. The utilization of this diagnostic PCR assay in analysis of field soil and carrot tissue might in the future be exploited to reduce the incidence of this serious carrot disease.     

Occurrence of <Emphasis Type="Italic">Rhexocercosporidium carotae</Emphasis> on cold stored carrot roots in the Netherlands

Winter carrot for the fresh market is an important cash crop for many organic arable farms in the Netherlands. In recent years carrot roots from cold stores have been affected by superficial dark brown to black spots. To gain insight into the pathogens causing the blemish and the effect of agronomic practices on their occurrence, surveys were carried out among crops harvested in 2001 and 2002. In addition carrots harvested in 2003 were screened for root spotting pathogens. Rhexocercosporidium carotae (syn. Acrothecium carotae and Pseudocercosporidium carotae) was the dominant pathogen in blackish spots on carrots harvested in 2001. On carrots harvested in 2002 and 2003 Alternaria radicina was detected more frequently. Multiple regression analysis indicated that a higher occurrence of the blemish may be linked with harvest conditions and presence of umbelliferous plants. The effect of the temperature on conidial germination, mycelial growth and pathogenicity of R. carotae was studied. The estimated optimum and maximum temperature for growth of R. carotae was 19 and 29°C, respectively. Inoculation experiments demonstrated that wounds are good invasion routes. Infection occurred at 3, 10 and 20°C, but not at 30°C. Penetration into wounds was greatest at 20°C.     

Real‐time variable‐rate herbicide application for weed control in carrots

A camera sensor for precision weed control in arable fields has been developed at the Leibniz Institute for Agricultural Engineering. For herbicide spraying in carrots, the sensor was positioned at the front three‐point linkage of a tractor and was operated between carrot rows. In field trials in 2 years, real‐time (online) technology in which sensing and spraying were performed in one step was evaluated in comparison with a conventional uniform spraying application. The spray volume was linearly adjusted to the camera‐detected weed coverage level from a minimum of 200 L ha^?1 if no weeds were present to a maximum of 400 L ha^?1. The herbicide savings were 30% in 2007 and 34%, 43% and 36% for each of three applications in 2010. There were no significant differences between the camera‐based and conventional spraying approaches with regard to yield parameters, total carrot weight and weight of marketable carrots. Regarding the weed control efficiency of the camera‐based spraying procedure in the 2010 experiment, no trend was observed between the weed coverage and the application rate of the previous spraying.     

Biological and genetic characterization of carrot red leaf virus and its associated virus/RNA isolated from carrots in Hokkaido,Japan

Carrot motley dwarf (CMD) is caused by mixed infection of carrot red leaf virus (CtRLV) with either carrot mottle virus (CMoV) or carrot mottle mimic virus, and additional infection with CtRLV-associated RNA (CtRLVaRNA). Here, the author investigated the viruses or virus-like RNA isolated from carrots with reddening symptoms in Hokkaido, the northern island of Japan. Three types of infections were mainly detected: single infection with CtRLV, which was most prevalent; double infection with CtRLV and CMoV; and triple infection with CtRLV, CMoV, and CtRLVaRNA. Fields with the three agents were severely affected, with diseased plants showing mottling, whereas in fields where disease incidence was low and sporadic, CtRLV was often found alone in plants with mild symptoms. Inoculation tests using carrot plants showed that CMoV enhanced disease severity, and the RNA accumulation of CtRLV. However, in the presence of CtRLVaRNA (+ CMoV), distinct symptoms such as systemic mottling and stunting developed, while the enhancement of CtRLV accumulation was abolished. These results imply that CtRLVaRNA (+ CMoV) antagonizes CtRLV despite its dependence on CtRLV for aphid transmission, and that mixed infection with CtRLVaRNA is involved in the development of the conspicuous mottling. All agents detected in Hokkaido were very similar to European and American isolates in terms of their genomic sequences and host range. This represents the first report of CMD in Japan, and provides further information on the genetic and biological properties of CMD-associated agents, as well as the aetiology of the disease.     

Integrated Management for Carrot Weevil

The carrot weevil, Listronotus oregonensis (LeConte) (Coleoptera: Curculionidae) is a major pest of carrots in northeastern North America. The females oviposit in the leaf petiole and the developing larvae make tunnels in the carrot roots. Exclusive reliance on chemical control and the poor control obtained, prompted, in the early 1980s, research on the ecology and physiology of the pest and its natural enemies. Carrot weevil females start their oviposition by 147±9DD_7°C but they oviposit only in carrots past the 4 true-leaf-stage. As a result, oviposition is delayed, and damage reduced, in late-sown carrots. Monitoring carrot weevil adults in the spring with either carrot root sections or wooden plate traps enables growers to apply control treatments only if the population density justifies it. Egg parasitoids of the genus Anaphes (Hymenoptera: Mymaridae) are the most important natural enemies and they account, in untreated plots, for up to 80% mortality. Their effectiveness could be improved by managing field borders to establish secondary host species. An IPM programme was developed in the 1980s based on the research results obtained and, through regular monitoring, has achieved a substantial decrease in pesticide use.     

Haplotypes of “<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum” suggest long-standing separation

Three haplotypes of the recently discovered bacterium species “Candidatus Liberibacter solanacearum” are described and related to geographic ranges. The first two are associated with Zebra Chip/Psyllid Yellows of potatoes and other solanaceous plants, vectored by the tomato/potato psyllid Bactericera cockerelli in North and Central America and New Zealand. The third is associated with diseased carrots in Finland and vectored by the carrot psyllid Trioza apicalis. The haplotypes are described by SNPs on the 16s rRNA, 16s/23s ISR and 50s rplJ and rplL ribosomal protein genes. These SNPs are inherited as a package across the three genes. Haplotype “a” has been found primarily from Honduras and Guatemala through western Mexico to Arizona and California, and in New Zealand. Haplotype “b” is currently known from eastern Mexico and northwards through Texas to south central Washington. These haplotypes show some range overlap in Texas, Kansas and Nebraska. The haplotypes are not yet known to elicit biological differences in the plant or insect hosts. These apparently stable haplotypes suggest separate bacterial populations of long standing.     

Susceptibility of wild carrot (Daucus carota ssp. carota) to Sclerotinia sclerotiorum

Sclerotinia soft rot, caused by Sclerotinia sclerotiorum, is a severe disease of cultivated carrots (Daucus carota ssp. sativus) in storage. It is not known whether Sclerotinia soft rot also affects wild carrots (D. carota ssp. carota), which hybridise and exchange genes, among them resistance genes, with the cultivated carrot. We investigated the susceptibility of wild carrots to S. sclerotiorum isolates from cultivated carrot under controlled and outdoor conditions. Inoculated roots from both wild and cultivated plants produced sclerotia and soft rot in a growth chamber test. Two isolates differed significantly in the ability to produce lesions and sclerotia on roots of both wild carrots and cv. Bolero. Flowering stems of wild carrots produced dry, pale lesions after inoculation with the pathogen, and above-ground plant weight was significantly reduced 4 weeks after inoculation in a greenhouse test. Wild and cultivar rosette plants died earlier and fewer plants survived when inoculated with the pathogen under outdoor test conditions. Cultivar plants died earlier than wild plants, but survived as frequently. Plants inoculated in the crown died earlier and at a lower frequency than plants inoculated on leaves. Wild carrots may thus serve as a host of S. sclerotiorum and thus eventually benefit from any uptake of resistance genes, among them transgenes, via introgression from cultivated carrots.     

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.     

Supervised control of the carrot fly in the Netherlands; insights from a dirty dataset1

‘De Groene Vlieg’ (the Green Fly) is an independent company offering various services concerning integrated pest management in open field agriculture in the Netherlands. One such service is the supervised control of carrot fly (Psila rosae). From 1993 to 2008 the company has monitored the carrot fly population on a total of nearly 65 000 hectares resulting in an extensive dataset. The data was not collected according to proper experimental methods, hence it is titled dirty, but the size of the dataset does allow valuable insights to be gained. De Groene Vlieg monitors the carrot fly population on each field separately. Sticky traps are collected weekly and flies are counted in the laboratory. Growers receive messages containing advice regarding when to apply insecticides and they are responsible for doing so. Overall, over a third of the fields require no spraying at all and for 75% of the fields growers are advised to spray three times or less. One insight from the dataset is that small carrots require more insecticide treatments than large carrots (‘winter carrots’). Reasons for this difference are probably both regional and cultural. Additionally, focusing on a small and uniform area reveals that carrot fly population dynamics are highly variable between years. Another insight is that sowing date has a large effect on the number of insecticide sprayings that are required. Fields sown before the first flight have more flies in the second flight than fields sown later. Comparison of biological and conventional carrot fields shows that carrot fly populations are similar for the first flight. However, during the second flight the number of flies is much higher on organic fields than on conventional ones. Finally, insecticide seed treatment with chlorfenvinphos to control the reproduction of the first flight appears slightly more efficient than supervised control. However, supervised control seems as efficient as seed treatment with clothianidin and beta cyfluthrin.     

Occurrence of Pythium Ultimum Var. Ultimum in a Greenhouse on Spitsbergen Island, Svalbard

Pythium ultimum var. ultimum was isolated from carrot seedlings with damping off and from soil used for growing the plant in a greenhouse on Spitsbergen Island, Svalbard. The fungus caused severe damping off of carrot, cucumber and tomato seedlings after artificial inoculation. The rDNA internal transcribed spacer sequences of the Svalbard isolate were identical to those of Canadian and Japanese isolates of P. ultimum var. ultimum. The results suggest that the pathogen in the greenhouse on Svalbard was probably introduced from temperate regions through contaminated plants and/or soil imported to the island. This is the first record of P. ultimum var. ultimum within the Arctic zone.     

Haplotypes of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’ identified in Umbeliferous crops in Spain

‘Candidatus Liberibacter solanacearum’ is a phloem-limited Gram-negative bacterium that causes serious damage to different crops of the botanical families Solanaceae and Apiaceae. Five haplotypes have been described: LsoA and LsoB are present in solanaceous crops in America and vectored by the tomato/potato psyllid Bactericera cockerelli; LsoC affects carrots from Northern and Central Europe, and is transmitted by the carrot psyllid Trioza apicalis; haplotypes LsoD and LsoE are present in Southern Europe and Morocco in carrot and celery, and are associated with the psyllid Bactericera trigonica. Thirty-four ‘Ca. L. solanacearum’ isolates were collected in six different regions of Spain from distinct Apiaceae hosts (carrot, celery, parsley and parsnip) in eight consecutive years and were analysed. Their haplotypes were determined by a sequence analysis of 16S ribosomal RNA, the 16S–26S ribosomal RNA intergenic spacer, and the 23S ribosomal RNA and rplJ and rplL genes. Both haplotypes LsoD and LsoE were found across Spain, and no host specificity appeared between these two haplotypes. This is the first report of ‘Ca. L. solanacearum’ associated with parsley and parsnip.     

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.     

Identification of a bacterium isolated from galls on carrot and weeds

In 2004, bacterial galls were found on the roots of carrots in Shizuoka Prefecture, Japan. Galls were about 0.1–2 cm in diameter, light brown in color and had rough surfaces. In 2005, similar galls were found on the roots of three weeds: henbit (Lamium amplexicaule L.), Persian speedwell (Veronica persica Poir.) and leaf mustard (Brassica juncea L.). A bacterium that forms white, rough colonies was isolated from the carrot and weeds galls. The bacterial isolates had properties identical with Rhizobacter dauci Goto and Kuwata. Phylogenetic analysis based on 16S rDNA sequences showed that the carrot isolate had the highest homology (similarity of 100%) with that of the type strain of R. dauci. Rep-PCR genomic fingerprinting using BOX A1R primer showed that the carrot and weeds isolates were nearly identical. Pathogenicity of the isolates was confirmed by inoculating the roots of carrots and the weeds. After 2–5 weeks, they formed galls on the roots of the original host species and on other plant species tested. The galls were indistinguishable from those formed naturally, and the inoculated bacterium was reisolated. Thus, the causal bacterium of carrot and weeds gall was identified as R. dauci, and the bacterium was found to have a wider host range than previously known. These weed hosts may serve as inoculum sources for carrot bacterial gall disease.     

Airborne basidiospores as an inoculum source of <Emphasis Type="Italic">Typhula variabilis</Emphasis> and the effect of hilling on the incidence of Typhula winter rot of carrots

Typhula winter rot on overwintering carrots caused by Typhula variabilis is a newly confirmed disease, and no practical control measure is yet available. To develop a control method, here we researched the infection period of T. variabilis and the time that winter rot appeared on carrots. Using spore traps, we found that basidiospore rain occurred from September to November before snowfall in Memuro, Hokkaido. In addition, carrot leaves collected in autumn had already been infected by T. variabilis. These epidemiological investigations revealed that the pathogen releases basidiospores to infect carrot leaves before snow cover, resulting in root decay under snow. An effective control method was then developed to avoid direct contact of basidiospores of T. variabilis with plant tops by covering the plants with soil in autumn. Thus, the percentage of rotted roots was reduced to about half.     

Occurrence of fungal pathogens of carrots on wooden boxes used for storage

Infested wooden boxes, previously used for carrot storage, were sampled in four commercial carrot production farms in Bradford Marsh, Ontario, and screened for fungal occurrence. At least 128 and 465 fungal isolates were recovered from these boxes in 2001 and 2002, respectively, and were classified into 10 taxonomic groups, including Alternaria spp., Aspergillus spp., Botrytis cinerea , Fusarium spp., Mucor spp., Penicillium spp., Rhizoctonia carotae , Rhizopus spp., Sclerotinia sclerotiorum and Trichoderma spp. A subsample of 27 putative pathogenic isolates was further tested for the ability to cause disease on carrots and to colonize wood surfaces under growth room and cold storage conditions. Approximately 60% of the taxa growing on wood caused lesions upon contact with intact carrots in cold storage. Isolates of S. sclerotiorum , B. cinerea and R. carotae caused the most severe diseases, developed most extensively on wooden surfaces in cold storage, and represented 12% of the recovered fungi. Isolates of Alternaria spp., Aspergillus spp., Fusarium spp., Mucor spp., Penicillium spp., Rhizopus spp. and Trichoderma spp. caused negligible or no disease on carrots and represented 88% of recovered fungi. Several of these fungi, however, showed potential to colonize wooden surfaces and cause disease on sliced carrots. This study suggests that pathogenic inocula occurring on used wooden boxes can initiate disease upon contact with healthy carrots and reusing infested boxes can affect carrots in storage.     

Distribution ofLeptosphaeria maculans in two fields in southern Ontario as determined by the polymerase chain reaction

The distribution of the highly virulent and weakly virulent types ofLeptosphaeria maculans, causal agent of blackleg of oilseed rape, was studied in two fields in southern Ontario. Using a polymerase chain reaction-based assay with primers specific for these virulence types, plant tissues were directly examined for the occurrence of the pathogen. The highly virulent type was detected in leaf, stem and crown tissue at most of the sampling sites. The weakly virulent type was detected only in leaf lesions at 50% of the sites in field 1 and 30% of the sites in field 2. Of 96 leaf lesions examined, 48 contained the highly virulent type, 12 contained the weakly virulent type and 16 contained both the highly virulent and weakly virulent types.Sclerotinia sclerotiorum was isolated from all leaf lesion that did not react withL. maculans virulence type-specific primers. Coinfection of single blackleg leaf lesions by bothL. maculans virulence types thus occurred in oilseed rape plants in the field. Only the highly virulent type was detected in pseudothecia on stubble. Approximately 1% of the seed collected from these two fields containedL. maculans, and both the highly virulent and weakly virulent types were detected. The highly virulent type was more prevalent and non-specific in the kind of plant tissue it infected, whereas the weakly virulent type appeared to be limited to infecting leaves and seed. This study illustrates an application of polymerase chain reaction with virulence type-specific oligonucleotide primers to study the epidemiology of blackleg of oilseed rape.     

Rotylenchus uniformis (Thorne) on carrots

Rotylenchus uniformis (Thorne) causes small orange lesions in the cortex of carrot roots and also orange discoloration of the stele in the vicinity of these lesions. The tolerance limit of carrots toR. uniformis was about 30 nematodes per 5 g soil in pot trials at about 17°C and 2 nematodes per 5g soil at 5° to 10°C. However, this temperature is too low for normal development of the plant. Field observations in the literature suggest that carrot yields are not reduced at densities ofR. uniformis up to 20 nematodes per 5 g soil. No field data are available for higher densities. From the frequency distribution of different densities ofR. uniformis in soil samples from carrot fields, and assuming a tolerance limit of 16 nematodes per 5 g soil, the area where losses occur is estimated to be between 10 and 15% of the total area under carrots. The total reduction of yield by the nematode would then be less than 1%. If the tolerance limit is 32 nematodes per 5 g soil, the damage would be negligible.Carrot, cauliflower andPhaseolus beans are among the best hosts ofR. uniformis.     

Pathogenicity of Colletotrichum gloeosporioides to carrot

Abstract

Colletotrichum gloeosporioides was identified as the causal agent of carrot umbel blight in Brazil. Pathogenicity was evaluated on carrot and other host plants, as well as compared with the pathogenicity of other C. gloeosporioides isolates on carrot. C. gloeosporioides isolated from carrot umbel was able to infect tomato plants and ripe fruits of tomato and sweet pepper, in addition to carrot umbels and seedlings. This appears to be the first report of C. gloeosporioides attacking carrots.