Transmission of ‘Candidatus Phytoplasma pyri’ by naturally infected Cacopsylla pyri to peach,an approach to the epidemiology of peach yellow leaf roll (PYLR) in Spain

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.     

Distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in European fruit‐growing areas: a review

European stone fruit yellows (ESFY) is an EU‐listed I/AII disease affecting Prunus spp. caused by ‘Candidatus Phytoplasma prunorum’. This paper reports the results from a systematic literature review approach that sought to determine the geographic distribution of ‘Ca. Phytoplasma prunorum’ in European fruit‐growing areas. Evidence for the presence of the phytoplasma was found for 15 of the 27 EU countries. It is prevalent in the most important stone fruit production areas of Central and Southern Europe, where it causes substantial impact in apricots (Prunus armeniaca), Japanese plums (P. salicina) and peaches (P. persica). In Northern European areas where these hosts are not produced, it is occasionally found on tolerant species (P. domestica). However, because surveys of the disease status of tolerant hosts are not performed, it remains unclear whether the pathogen is absent in Northern Europe or survives in tolerant cultivated or wild hosts. No reports of ESFY were found from the southernmost part of Europe: Portugal, Spain (Andalucia, Castile–La Mancha), Italy (Sicily, Puglia), Greece (Crete), Cyprus and Malta. This may be explained by the absence of the favoured wild hosts of the vector. Moreover, it remains unclear if the vector finds suitable conditions for aestivation and overwintering in these regions.     

The insect vector Cacopsylla picta vertically transmits the bacterium ‘Candidatus Phytoplasma mali’ to its progeny

The phloem‐sucking psyllid Cacopsylla picta plays an important role in transmitting the bacterium ‘Candidatus Phytoplasma mali’, the agent associated with apple proliferation disease. The psyllid can ingest ‘Ca. Phytoplasma mali’ from infected apple trees and spread the bacterium by subsequently feeding on uninfected trees. Until now, this has been the most important method of ‘Ca. Phytoplasma mali’ transmission. The aim of this study was to investigate whether infected C. picta are able to transmit ‘Ca. Phytoplasma mali’ directly to their progeny. This method of transmission would allow the bacteria to bypass a time‐consuming reproductive cycle in the host plant. Furthermore, this would cause a high number of infected F₁ individuals in the vector population. To address this question, eggs, nymphs and adults derived from infected overwintering adults of C. picta were reared on non‐infected apple saplings and subsequently tested for the presence of ‘Ca. Phytoplasma mali’. In this study it was shown for the first time that infected C. picta individuals transmit ‘Ca. Phytoplasma mali’ to their eggs, nymphs and F₁ adults, thus providing the basis for a more detailed understanding of ‘Ca. Phytoplasma mali’ transmission by C. picta.     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.     

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.     

Frequency and occurrence of the carrot pathogen ‘Candidatus Liberibacter solanacearum’ haplotype C in Finland

Occurrence of ‘Candidatus Liberibacter solanacearum’ (CLso) was studied in field‐grown carrots (Daucus carota) in different regions of Finland. In addition, the frequency of CLso in carrots and in field populations of its vector, the carrot psyllid (Trioza apicalis), was studied in southwestern Finland. CLso was detected in six of the seven regions where the main carrot cultivation areas are located. The highest disease incidence was found in southwestern Finland, in the area where this carrot pathogen was originally found. In the Tavastia Proper and Southwest Finland regions, CLso was detected in 26 out of 30 randomly chosen fields inspected in 2013 and 2014, and in a third of those fields more than 10% of plants showed symptoms. Of those carrots showing both psyllid feeding‐associated leaf curling and CLso infection‐associated leaf discolouration symptoms, 77% were CLso positive in the PCR test. Some symptomless carrots from the affected fields also tested positive. Of the carrot psyllid individuals collected from the same area, 60% were CLso positive. Elsewhere, disease incidence was variable in South Ostrobothnia in western Finland and low but established in South Savonia in eastern Finland. CLso was not detected in the North Ostrobothnia region. Sequencing of the amplified DNA fragments confirmed that the bacteria in the carrot samples from different areas within Finland all represented CLso haplotype C. The frequent occurrence and wide distribution of this pathogen, transmitted by a psyllid that does not migrate over long distances, suggest that it is persistent in Finland.     

A yellows disease system with differing principal host plants for the obligatory pathogen and its vector

A stolbur‐type phytoplasma is the putative pathogen of grapevine yellows disease that causes economic damage to vineyards in most growing areas around the world. The pathogen is known to be transmitted to vines by two planthoppers, Hyalesthes obsoletus and Reptalus panzer; the latter is found in Europe but has not yet been observed in Israel. The establishment of a vector–pathogen–plant relationship requires that the pathogen and the vector meet on a shared host plant. This does not happen in the ecosystem examined here, where two different principal host plants for the obligate pathogen and its vector exist: the pathogen is established on vines, while its vector, H. obsoletus, develops on Vitex agnus‐castus. The present study verified that: (i) the vector cannot complete its life cycle on vines; (ii) V. agnus‐castus does not grow in the immediate vicinity of vines, and does not harbour the pathogen; and (iii) the pathogen is not vertically transmitted from mother to offspring. Moreover, in a thorough search of plants in vine growing areas, no other plants were found that host both the vector and the pathogen. However, it was found that the planthopper can acquire the phytoplasma from infected vines. Nonetheless, this does not prove the ability of the planthopper to further transmit the pathogen to vines and does not explain the presence of the vector on the non‐preferred vines. Thus, the enigma of the pathogen–vector–host triangle in this system remains unresolved.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Molecular tracing of the transmission routes of bois noir in Mediterranean vineyards of Montenegro and experimental evidence for the epidemiological role of Vitex agnus‐castus (Lamiaceae) and associated Hyalesthes obsoletus (Cixiidae)

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.     

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.     

Isolation and characterization of novel soilborne lytic bacteriophages infecting Dickeya spp. biovar 3 (‘D. solani’)

Nine bacteriophages infecting Dickeya spp. biovar 3 (‘Dickeya solani’) were isolated from soil samples collected in different regions in Poland. The phages have a typical morphology of the members of the order Caudovirales, family Myoviridae, with a head diameter of c. 90–100 nm and tail length of c. 120–140 nm. In host range experiments, phage ?D5 expressed the broadest host range, infecting members of all Dickeya spp., and phage ?D7 showed the narrowest host range, infecting isolates of Dickeya dadantii and ‘D. solani’ only. None of the phages was able to infect Pectobacterium spp. isolates. All phages were prone to inactivation by pH 2, temperature of 85°C and by UV illumination for 10 min (50 mJ cm^?2). Additionally, phages ?D1, ?D10 and ?D11 were inactivated by 5 m NaCl and phage ?D2 was inactivated by chloroform. Phages ?D1, ?D5, ?D7 and ?D10 were characterized for optimal multiplicity of infection and the rate of adsorption to the bacterial cells. The latent period was 30 min for ?D1, 40 min for ?D5, 20–30 min for ?D7 and 40 min for ?D10. The estimated burst size was c. 100 plaque‐forming units per infected cell. The bacteriophages were able to completely stop the growth of ‘D. solani’ in vitro and to protect potato tuber tissue from maceration caused by the bacteria. The potential use of bacteriophages for the biocontrol of biovar 3 Dickeya spp. in potato is discussed.     

Up‐regulation of PR1 and less disruption of hormone and sucrose metabolism in roots is associated with lower susceptibility to ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las), is a devastating disease of citrus trees in Florida. Previous work showed that the rootstock cultivar Cleopatra mandarin (Citrus reticulata) has a higher population of Las in roots than Swingle citrumelo (C. paradisi × Poncirus trifoliata). Las reduced fibrous root biomass and sucrose content in Cleopatra mandarin more than in Swingle citrumelo. To understand the mechanisms for susceptibility to Las infection, sucrose and hormone metabolism status were evaluated in Cleopatra mandarin and Swingle citrumelo. In fibrous roots of Cleopatra mandarin, higher expression of genes related to sucrose cleavage was consistent with lower sucrose content compared to noninoculated seedlings at 5 weeks post‐root trimming (wpt). In fibrous roots of Swingle citrumelo, both sucrose content and gene expression related to sucrose cleavage were less disrupted by Las infection compared to Cleopatra mandarin at 5 wpt. Genes associated with salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) synthesis, and ABA signalling, phospholipases D (PLD), and phospholipase A2 (PLA2) were activated by Las infection at 5 wpt in Cleopatra mandarin. Expression of downstream effectors of SA, i.e. NPR1, WRKY70 and PR1, did not change in Cleopatra mandarin, suggesting inhibition of the response to SA by the elevation of ABA, ET and PLD. In contrast, the up‐regulation of PR1, lower response of sucrose metabolism genes and down‐regulation of biosynthesis of phytohormones indicates that Swingle citrumelo activates a more effective defence against this biotrophic pathogen than Cleopatra mandarin.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

Non‐pathogenic rhizosphere bacteria belonging to the genera Rhizorhapis and Sphingobium provide specific control of lettuce corky root disease caused by species of the same bacterial genera

Lettuce corky root (CR) is caused by bacteria in the genera Rhizorhapis, Sphingobium, Sphingopyxis and Rhizorhabdus of the family Sphingomonadaceae. Members of this family are common rhizosphere bacteria, some pathogenic to lettuce. Sixty‐eight non‐pathogenic isolates of bacteria obtained from lettuce roots were tested for control of CR caused by Rhizorhapis suberifaciens CA1^T and FL1, and Sphingobium mellinum WI4^T. In two initial screenings, 10 isolates significantly reduced CR induced by one or more pathogenic strains on lettuce seedlings in vermiculite, while seven non‐pathogenic isolates provided significant CR control in natural or sterilized field soil. Rhizorhapis suberifaciens FL11 was effective at controlling all pathogenic strains, but most effective against R. suberifaciens CA1^T. The other selected isolates controlled only pathogenic strains belonging to their own genus. In a greenhouse experiment, a soil drench with selected biocontrol agents (R. suberifaciens FL11, Sphingomonas sp. NY3 and S. mellinum CA16) controlled CR better than seed treatments or application of alginate pellets. In microplots infested with R. suberifaciens CA1^T, seed treatment with R. suberifaciens FL11 provided complete control and a soil drench with FL11 significantly reduced the disease. Pathogenicity tests with FL11 on 23 plant species in 10 families resulted in slight yellowing on roots of lettuce and close relatives; similar yellowing appeared on some roots of non‐inoculated lettuce plants. This research showed that biocontrol agents can be genus‐specific. Only one isolate, FL11, provided more general control of various pathogenic strains causing CR even in field soil in pots and microplots.     

Between‐site and ‐year variation in the relative susceptibility of native Scottish Pinus sylvestris populations to dothistroma needle blight

Dothistroma needle blight (DNB), caused by Dothistroma septosporum, is currently the disease causing most concern in British pine plantations. Previous artificial inoculation (AI) experiments showed that native Scottish Scots pine (Pinus sylvestris) populations vary in susceptibility to DNB. However, it is unclear if the relative susceptibility of Scots pine populations observed in these experiments can be replicated under natural conditions. It is also unknown whether relative susceptibility of Scots pine populations varies between sites or years. To answer these two questions, young Scots pine plants from six native Scottish populations (Abernethy, Allt Broighleachan, Amat, Beinn Eighe, Glen Cannich and Glen Loyne) were exposed to natural D. septosporum inoculum at two Scottish sites (Culbin and Torrs Warren) between 2012 and 2014. DNB disease incidence and severity was assessed each October. Relative susceptibilities of the Scots pine populations varied between sites and across years. In two of the three years at Torrs Warren (2012 and 2014), the relative susceptibilities of the populations were strongly positively correlated with those observed in previous AI experiments. In these years, trees from Glen Loyne and Glen Cannich were the most susceptible. Conversely, there was no correlation between the relative susceptibilities seen in any year at Culbin with those observed in AI experiments. At Culbin, Beinn Eighe was the most susceptible population. Across both sites, there was a strong positive relationship between total summer precipitation and DNB severity (R = 0·93, t = 8·2, P = 0·001).