Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

Phytoplasma Transmission by Heterologous Grafting Influences Viability of the Scion and Results in Early Symptom Development in Periwinkle Rootstock

The stolbur phytoplasma ‘Candidatus Phytoplasma solani’ is responsible for the grapevine disease ‘bois noir’ affecting a number of wine‐growing areas in Europe. Transmission of stolbur phytoplasma to different laboratory hosts can be difficult due to the requirement of transmitting insect vectors or parasite plants. Here, heterologous grafting was used as an alternative technique for transmission of common and strongly symptomatic stolbur genotypes CPsM4_At1 and CPsM4_At6 of ‘Ca. P. solani’ to experimental host plants such as Catharanthus roseus and tomato making phytoplasma strains more accessible for molecular and experimental investigations in different plant species. Transmission was confirmed by quantitative PCR, microscopy and nested PCR followed by marker gene sequencing. In our study, the transmission of different genotypes of ‘Ca. P. solani’ resulted in distinguishable symptom development in the laboratory host C. roseus. Symptom development in grafted rootstock was observed three to 7 weeks after heterologous grafting. Survival of the graft unit was influenced by the presence of ‘Ca. P. solani’ in the scions and was clearly reduced in phytoplasma free scion – rootstock combinations.     

Biochemical and epigenetic changes in phytoplasma‐recovered periwinkle after indole‐3‐butyric acid treatment

Aim: To elucidate the possible mechanism of phytoplasma elimination from periwinkle shoots caused by indole‐3‐butyric acid (IBA) treatment. Methods and Results: It has been shown that a transfer of in vitro‐grown phytoplasma‐infected Catharanthus roseus (periwinkle) plantlets from medium supplemented with 6‐benzylaminopurine (BA) to one supplemented with IBA can induce remission of symptoms and even permanent elimination of ‘Candidatus Phytoplasma asteris’ reference strain HYDB. Endogenous auxin levels and general methylation levels in noninfected periwinkles, periwinkles infected with two ‘Candidatus Phytoplasma’ species and phytoplasma‐recovered periwinkles were measured and compared. After the transfer from cytokinin‐ to auxin‐containing media, healthy shoots maintained their phenotype, methylation levels and hormone concentrations. Phytoplasma infection caused a change in the endogenous indole‐3‐acetic acid to IBA ratio in periwinkle shoots infected with two ‘Candidatus Phytoplasma’ species, but general methylation was significantly changed only in shoots infected with ‘Ca. P. asteris’, which resulted in the only phytoplasma species eliminated from shoots after transfer to IBA‐containing medium. Both phytoplasma infection and treatment with plant growth regulators influenced callose deposition in phloem tissue, concentrations of photosynthetic pigments and soluble proteins, H₂O₂ levels and activities of catalase (CAT) and ascorbate peroxidase (APX). Conclusion: Lower level of host genome methylation in ‘Ca. P. asteris’‐infected periwinkles on medium supplemented with BA was significantly elevated after IBA treatment, while IBA treatment had no effect on cytosine methylation in periwinkles infected with ‘Candidatus Phytoplasma ulmi’ strain EY‐C. Significance and Impact of the Study: Hormone‐dependent recovery is a distinct phenomenon from natural recovery. As opposed to spontaneously recovered plants in which elevated peroxide levels and differential expression of peroxide‐related enzymes were observed, in hormone‐dependent recovery changes in global host genome, methylation coincide with the presence/absence of phytoplasma.     

Phytoplasma from little leaf disease affected sweetpotato in Western Australia: detection and phylogeny

Symptoms of leaf and stem chlorosis and plant stunting were common in sweetpotato plants (Ipomoea batatas) in farmers’ fields in two widely separated locations, Kununurra and Broome, in the tropical Kimberley region in the state of Western Australia in 2003 and 2004. In the glasshouse, progeny plants developed similar symptoms characteristic of phytoplasma infection, consisting of chlorosis and a stunted, bushy appearance as a result of proliferation of axillary shoots. The same symptoms were reproduced in the African sweetpotato cv. Tanzania grafted with scions from the plant Aus1 with symptoms and in which no viruses were detected. PCR amplification with phytoplasma‐specific primers and sequencing of the 16S‐23S rRNA gene region from two plants with symptoms, Aus1 (Broome) and Aus142A (Kununurra), revealed highly identical sequences. Phylogenetic analysis of the 16S rRNA gene sequences obtained from previously described sweetpotato phytoplasma and inclusion of other selected phytoplasma for comparison indicated that Aus1 and Aus142A belonged to the Candidatus Phytoplasma aurantifolia species (16SrII). The 16S genes of Aus1 and Aus142A were almost identical to those of sweet potato little leaf (SPLL‐V4) phytoplasma from Australia (99.3%–99.4%) but different from those of the sweetpotato phytoplasma from Taiwan (95.5%–95.6%) and Uganda (SPLL‐UG, 90.0%–90.1%). Phylogenetically, Aus1, Aus142A and a phytoplasma previously described from sweetpotato in the Northern Territory of Australia formed a group distinctly different from other isolates within Ca. Phytoplasma aurantifolia species. These findings indicate that novel isolates of the 16SrII‐type phytoplasma pose a potential threat to sustainable sweetpotato production in northern Australia.     

Development of a new probe for specific and sensitive detection of 'Candidatus Phytoplasma mali' in inoculated apple trees

A new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of ‘Candidatus Phytoplasma mali’. The new probe can distinguish a single mismatch between ‘Ca. P. mali’ and ‘Candidatus Phytoplasma prunorum’, this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real‐time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real‐time PCR method, three other real‐time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real‐time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our real‐time PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.     

Association of two groups of phytoplasma with various symptoms in some wooden and herbaceous plants

During 2015–2016, wooden and herbaceous plants growing in parks, boulevards, fields, gardens and forests in Khuzestan province, southwestern Iran, were visually inspected for symptoms resembling phytoplasma. Fifty‐one symptomatic samples from nine different species and one symptomless sample from each plant were collected. Leaf midribs, petioles and the parts of stem cambium were separated and freeze‐dried. Total DNA was extracted using CTAB‐based method and tested for phytoplasma using a nested PCR assay. The expected size amplicons of 16S rDNA were sequenced and compared to those of reference phytoplasmas by BLASTn search and phylogenetic analysis. The consensus 16S rDNA sequence of the detected phytoplasma in narrow cattail related to reference phytoplasma group 16SrVI, “Candidatus Phytoplasma trifolii” while in the other plants were related to reference phytoplasma subgroup 16SrII–D, “Candidatus Phytoplasma aurantifolia.” All isolates showed 98%–99% sequence identity to members of their reference groups. To our knowledge, this is the first report of “Candidatus Phytoplasma aurantifolia”‐related strains infecting the plants of Acacia salicina, Alternanthera ficoidea, Melaleuca citrine, Citrus aurantium throughout the world and Celosia christata in Iran. Furthermore, this study is the first to report the association of a “Candidatus Phytoplasma trifolii”‐related strain with Typha angustifolia worldwide.     

Transcriptomics assisted proteomic analysis of Nicotiana occidentalis infected by Candidatus Phytoplasma mali strain AT

Phytoplasmas are pathogenic bacteria within the class of Mollicutes, which are associated with more than 1000 plant diseases. In this study, we applied quantitative mass spectrometry to analyse affected pathways of the model plant tobacco (Nicotiana occidentalis) upon Candidatus Phytoplasma mali strain AT infection. Using tissue obtained from leaf midribs, 1466 plant‐assigned proteins were identified. For 1019 of these proteins, we could reproducibly quantify the expression changes of infected versus noninfected plants, of which 157 proteins were up‐ and 173 proteins were downregulated. Differential expression took place in a number of pathways, among others strong downregulation of porphyrin and chlorophyll metabolism and upregulation of alpha‐linolenic acid metabolism, which was consistent with observed increased levels of jasmonic acid, a key signal molecule of plant defence. Our data shed light on the molecular networks that are involved in defence of plants against phytoplasma infection and provide a resource for further studies.     

Detection and Characterization of Phytoplasmas Infecting Ornamental and Weed Plants in Iran

During field surveys in 2004, ornamental and weed plants showing symptoms resembling those caused by phytoplasmas were observed in Mahallat (central Iran). These plants were examined for phytoplasma infections by polymerase chain reaction (PCR) assays using universal phytoplasma primers directed to ribosomal DNA (rDNA). All affected plants gave positive results. The detected phytoplasmas were characterized and differentiated through restriction fragment length polymorphism (RFLP) and sequence analysis of PCR‐amplified rDNA. The phytoplasmas detected in diseased Asclepias curassavica and Celosia argentea were identified as members of clover proliferation phytoplasma group (16SrVI group) whereas those from the remaining plants examined proved to be members of aster yellow phytoplasma group (16SrI group) (‘Candidatus Phytoplasma asteris’). In particular, following digestion with AluI, HaeIII and HhaI endonucleases, the phytoplasma detected in Limonium sinuatum showed restriction profiles identical to subgroup 16SrI‐C; phytoplasmas from Gomphocarpus physocarpus, Tanatacetum partenium, Lactuca serriola, Tagetes patula and Coreopsis lanceolata had the same restriction profiles as subgroup 16SrI‐B whereas Catharanthus roseus‐ and Rudbeckia hirta‐infecting phytoplasmas showed restriction patterns of subgroup 16SrI‐A. This is the first report on the occurrence of phytoplasma diseases of ornamental plants in Iran.     

Surveys reveal a complex association of phytoplasmas and viruses with the blueberry stunt disease on Canadian blueberry farms

Surveys for phytoplasmas and viruses were conducted during September 2014 and 2015 on highbush blueberry farms in the Région Montérégie, Quebec. Total DNA and RNA were extracted from blueberry bushes showing blueberry stunt (BBS) symptoms and from symptomless blueberry bushes, and utilised as templates for PCR and RT‐PCR assays, respectively. Phytoplasma DNA was amplified with universal phytoplasma primers that target the 16S rRNA, secA and secY genes from 12 out of 40 (30%) plants tested. Based on 16S rRNA, secA and secY gene sequence identity, phylogenetic clustering, actual and in silico RFLP analyses, phytoplasma strains associated with BBS disease in Quebec were identified as ‘Candidatus Phytoplasma asteris’‐related strains, closely related to the BBS Michigan phytoplasma strain (16SrI‐E). The secY gene sequence‐based single nucleotide polymorphism analysis revealed that one of the BBS phytoplasma strains associated with a leaf marginal yellowing is a secY‐I RFLP variant of the subgroup 16SrI‐E. Two viruses were detected in blueberry bushes. The Blueberry Red Ringspot Virus (BRRV) was found in a single infection in the cultivar Bluecrop with no apparent typical BRRV symptoms. The Tobacco Ringspot Virus (TRSV) was found singly infecting blueberry plants and co‐infecting a BBS phytoplasma‐infected blueberry cv. Bluecrop plant. This is the first report of TRSV in the cv. Bluecrop in Quebec. The Quebec BBS phytoplasma strain was identified in the leafhopper Graphocephala fennahi, which suggests that G. fennahi may be a potential vector for the BBS phytoplasma. The BBS disease shows a complex aetiology and epidemiology; therefore, prompt actions must be developed to support focused BBS integrated management strategies.     

Molecular biology and pathogenicity of phytoplasmas

Phytoplasmas are a large group of plant‐pathogenic wall‐less, non‐helical, bacteria associated with diseases, collectively referred to as yellows diseases, in more than a thousand plant species worldwide. Many of these diseases are of great economic importance. Phytoplasmas are difficult to study, in particular because all attempts at culturing these plant pathogens under axenic conditions have failed. With the introduction of molecular methods into phytoplasmology about two decades ago, the genetic diversity of phytoplasmas could be elucidated and a system for their taxonomic classification based on phylogenetic traits established. In addition, a wealth of information was generated on phytoplasma ecology and genomics, phytoplasma–plant host interactions and phytoplasma–insect vector relationships. Taxonomically, phytoplasmas are placed in the class Mollicutes, closely related to acholeplasmas, and are currently classified within the provisional genus ‘Candidatus Phytoplasma’ based primarily on 16S rDNA sequence analysis. Phytoplasmas are characterised by a small genome. The sizes vary considerably, ranging from 530 to 1350 kilobases (kb), with overlapping values between the various taxonomic groups and subgroups, resembling in this respect the culturable mollicutes. The smallest chromosome, about 530 kb, is known to occur in the Bermuda grass white leaf agent ‘Ca. Phytoplasma cynodontis’. This value represents the smallest mollicute chromosome reported to date. In diseased plants, phytoplasmas reside almost exclusively in the phloem sieve tube elements and are transmitted from plant to plant by phloem‐feeding homopteran insects, mainly leafhoppers and planthoppers, and less frequently psyllids. Most of the phytoplasma host plants are angiosperms in which a wide range of specific and non‐specific symptoms are induced. Phytoplasmas have a unique and complex life cycle that involves colonisation of different environments, the plant phloem and various organs of the insect vectors. Furthermore, many phytoplasmas have an extremely wide plant host range. The dynamic architecture of phytoplasma genomes, due to the occurrence of repetitive elements of various types, may account for variation in their genome size and adaptation of phytoplasmas to the diverse environments of their plant and insect hosts. The availability of five complete phytoplasma genome sequences has made it possible to identify a considerable number of genes that are likely to play major roles in phytoplasma–host interactions. Among these, there are genes encoding surface membrane proteins and effector proteins. Also, it has been shown that phytoplasmas dramatically alter their gene expression upon switching between plant and insect hosts.     

Auxin influences symptom expression and phytoplasma colonisation in periwinkle infected with periwinkle leaf yellowing phytoplasma

Auxin imbalance was suggested as a key factor in phytoplasma symptom development. Furthermore, remission of the symptoms of phytoplasma‐infected shoots can be promoted by culturing them in vitro in high‐auxin‐containing media. Therefore, effect of spraying 1‐naphthaleneacetic acid (NAA) on infected periwinkle (Catharanthus roseus) with periwinkle leaf yellowing (PLY) phytoplasma was examined. 1‐Naphthaleneacetic acid stimulated symptom development in phytoplasma‐inoculated shoots. Accelerated symptom development was associated with early accumulation of phytoplasmas. Two PATHOGENESIS‐RELATED (PR) genes, CrPR1a and CrPR1b, were induced by PLY phytoplasma infection, and the induction was suppressed by NAA. Therefore, the accelerated symptom development may be due to the suppression effect of NAA on PR‐related defence. However, while NAA promoted symptom development on shoots inoculated with phytoplasma, more non‐symptomatic shoots containing no phytoplasma were observed, suggesting that NAA prevents phytoplasma colonisation in non‐symptomatic shoots. The expression of two genes encoding jasmonic acid (JA) biosynthesis key enzymes, lipoxygenase and allene oxide cyclase, was downregulated in non‐symptomatic shoots of infected plants, and remained downregulated after auxin treatment. Therefore, the auxin‐promoted resistance should be JA independent. Because auxin may promote symptom development of PLY phytoplasma‐infected periwinkles, it may not link to plant resistance to phytoplasma infection.     

Cyclamen (Cyclamen persicum L.): a dead-end host species for 16Sr-IB and -IC subgroup phytoplasmas

Phytoplasmas belonging to the 16S rDNA subgroups IB and IC were found in five cyclamen (Cyclamen persicum L.) plants showing virescence and yellow stunted leaves and one plant showing phyllody, rolled and thickened leaves, respectively. Two cyclamens, representing the two syndromes, were chosen as source plants for transmission trials in which three leafhopper species, known as vectors of IB and IC subgroup phytoplasmas, were used to inoculate cyclamen and periwinkle [Catharanthus roseus (L.) G. Don] test plants. Out of 366 tested plants only one periwinkle exposed to Euscelis incisus was found harbouring a 16Sr‐IB phytoplasma. Out of 60 tested vector insects, only one adult of Macrosteles quadripunctulatus and two of E. incisus fed on 16Sr‐IB source cyclamen gave a positive amplification signal in nested PCR. This extremely low level of transmission to both cyclamen and the very susceptible periwinkle strongly suggests that cyclamen, commonly found infected in crops, is an unsuitable species for phytoplasma acquisition and can be regarded as a dead‐end host plant for phytoplasmas belonging to both IB and IC subgroups. Indications for glasshouse management are drawn from these findings. Among the leafhoppers investigated E. incisus falls most under suspicion since it feeds better than the others on cyclamen, was able to transmit the disease to one periwinkle plant, and IB phytoplasmas were detected in two individuals.     

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

Symptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.     

Extended larval development time for aphid parasitoids in the presence of plant endosymbionts

Abstract 1. Variation in plant chemistry does not only mediate interactions between plants and herbivores but also those between herbivores and their natural enemies, and plants and natural enemies. 2. Endophytic fungi complete their whole life cycle within the host plant’s tissue and are associated with a large diversity of plant species. Endophytes of the genus Neotyphodium alter the chemistry of the host plant by producing herbivore toxic alkaloids. 3. Here we asked whether the endophyte‐tolerant aphid species Metopolophium festucae could be defended against its parasitoid Aphidius ervi when feeding on endophyte‐infected plants. In a laboratory experiment, we compared life‐history traits of A. ervi when exposed to hosts on endophyte‐infected or endophyte‐free Lolium perenne. 4. The presence of endophytes significantly increased larval and pupal development times, but did not affect the mortality of immature parasitoids or the longevity of the adults. Although the number of parasitoid mummies tended to be reduced on endophyte‐infected plants, the number of emerging parasitoids did not differ significantly between the two treatments. 5. This shows that the metabolism of individual aphids feeding on infected plants may be changed and help in the defence against parasitoids. An increase in parasitoid development time should ultimately reduce the population growth of A. ervi. Therefore, endophyte presence may represent an advantage for endophyte‐tolerant aphid species through extended parasitoid development and its effect on parasitoid population dynamics.     

Variation in vector competency depends on chrysanthemum yellows phytoplasma distribution within Euscelidius variegatus

Phytoplasmas are plant‐pathogenic Mollicutes transmitted by leafhoppers, planthoppers, and psyllids in a persistent propagative manner. Chrysanthemum yellows phytoplasma (CY) is a member of ‘Candidatus Phytoplasma asteris’, 16Sr‐IB, and is transmitted by at least three leafhopper species, Macrosteles quadripunctulatus Kirschbaum, Euscelidius variegatus Kirschbaum, and Euscelis incisus Kirschbaum (all Homoptera: Cicadellidae: Deltocephalinae). Although M. quadripunctulatus transmits CY with very high efficiency (near 100%), 25% of E. variegatus repeatedly fail to transmit CY. The aims of this work were to correlate vector ability with different pathogen distribution in the insect body and to investigate the role of midgut and salivary glands as barriers to CY transmission. Euscelidius variegatus individuals acquired CY by feeding on infected plants or by abdominal microinjection of a phytoplasma‐enriched suspension. Insects were individually tested for transmission on daisy seedlings [Chrysanthemum carinatum Schousboe (Asteraceae)], and thereafter analysed by real‐time polymerase chain reaction (PCR) for CY concentration on whole insects or separately on heads and the rest of the body. Hoppers were classified as early and late transmitters or non‐transmitters, according to the time inoculated plants required for expression of CY symptoms. Similar transmission efficiencies were achieved following feeding or abdominal microinjection, suggesting that salivary glands may be a major barrier to transmission. Following acquisition from infected plants, all transmitters tested positive by PCR, and 60% of non‐transmitters also tested positive although with a significantly lower CY concentration. This indicates that a minimum number of phytoplasma cells may be required for successful transmission. The midgut may have prevented phytoplasma entry into the haemocoel of PCR‐negative non‐transmitters. Results suggest that both midgut and salivary glands may act as barriers. To assess the effect on CY transmission of a specific parasitic bacterium of E. variegatus, tentatively named BEV (Bacterium Euscelidius variegatus), we established a BEV‐infected population by abdominal microinjection of BEV bacteria. The presence of BEV did not significantly alter the efficiency of CY transmission.     

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector,phyllogen, induces phyllody

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant‐pathogenic bacteria that transform plant floral organs into leaf‐like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma‐secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma‐infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two‐hybrid and in planta transient co‐expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin–proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody‐inducing gene family of ‘phyllogens’.     

The plant hormone salicylic acid interacts with the mechanism of anti‐herbivory conferred by fungal endophytes in grasses

The plant hormone salicylic acid (SA) is recognized as an effective defence against biotrophic pathogens, but its role as regulator of beneficial plant symbionts has received little attention. We studied the relationship between the SA hormone and leaf fungal endophytes on herbivore defences in symbiotic grasses. We hypothesize that the SA exposure suppresses the endophyte reducing the fungal‐produced alkaloids. Because of the role that alkaloids play in anti‐herbivore defences, any reduction in their production should make host plants more susceptible to herbivores. Lolium multiflorum plants symbiotic and nonsymbiotic with the endophyte Epichloë occultans were exposed to SA followed by a challenge with the aphid Rhopalosiphum padi. We measured the level of plant resistance to aphids, and the defences conferred by endophytes and host plants. Symbiotic plants had lower concentrations of SA than did the nonsymbiotic counterparts. Consistent with our prediction, the hormonal treatment reduced the concentration of loline alkaloids (i.e., N‐formyllolines and N‐acetylnorlolines) and consequently decreased the endophyte‐conferred resistance against aphids. Our study highlights the importance of the interaction between the plant immune system and endophytes for the stability of the defensive mutualism. Our results indicate that the SA plays a critical role in regulating the endophyte‐conferred resistance against herbivores.     

Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.     

Molecular Detection and Identification of a 16SrVI Group Phytoplasma Associated with Tomato Big Bud Disease in Xinjiang,China

In 2010, tomato plants with big bud symptoms were observed in Xinjiang, China. PCR products of approximately 1.2 and 2.8 kb were amplified from infected tomato tissues but not from asymptomatic plants. A comparison of 16S rDNA sequences showed that the casual tomato big bud (TBB) phytoplasma was closely (99%) related to the ‘Candidatus Phytoplasma trifolii’ (16SrVI group). The TBB phytoplasma clustered into one branch with the Loofah witches'‐broom phytoplasma according to the 23S rDNA analysis but with no other member of the 16SrVI group. The cause of TBB symptoms was identified as ‘Ca. Phytoplasma trifolii' (16SrVI group) by PCR, virtual RFLP and sequencing analyses. This is the first report of a phytoplasma related to ‘Ca. Phytoplasma trifolii' causing TBB disease in China.