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During survey from 2013 to 2018, Cynodon dactylon at Shahjahnapur, Azamagarh, Oplismenus burmannii at Shahjahnapur, Arundo donax at Deoria and Ocimum cannum at Gorakhpur were observed with phytoplasma suspected symptoms. The maximum disease incidence (34%) was recorded at Deoria District in case of A. donax plant with witches' broom symptom. The nested PCR using universal primer pairs (R16F2n/R16R2) confirmed the phytoplasma association with all the suspected samples. Further BLASTn and phylogenetic analysis of the sequences revealed the association of 16SrXIV-A subgroups phytoplasma with C. dactylon, O. burmannii and phytoplasma belonging to 16SrI-B subgroups with A. donax and O. cannum plants in the present study.

Severe little leaf and excessive shoot proliferation symptoms typical of the peanut little leaf (PnLL) disease were observed in different peanut (Arachis hypogaea) fields, variety K-6, at Kadiriand Gooty regions, of Andhra Pradesh, India, during the monsoon season of 2018–19. Disease incidence was 2–3%. The phytoplasma etiology of the recorded disease was confirmed by amplifying ~1.25 kb DNA products of 16S rRNA gene from symptomatic plants using nested polymerase chain reaction with phytoplasma specific primers, P1/P7 and R16F2n/R16R2, respectively. A further confirmation of the phytoplasma etiology was obtained by amplification of 840 bp, 1094 bp and 465 bp DNA fragments using phytoplasma specific primers targeting secA, tuf and SAP11 genes, respectively. Sequence and phylogenetic analyses of the amplified 16S rRNA, secA, tuf and SAP11 genes revealed that the detected phytoplasma is a member of the peanut witches’-broom phytoplasma group or 16SrII group (‘Candidatus Phytoplasma australasia’). Also, on the basis of computer-simulated RFLP (= in silico RFLP) analysis of amplified 16S rRNA gene, the detected phytoplasma was assigned to subgroup D (16SrII-D). Parthenium hysterophorus plants showing witches’ broom and Cleome viscosa plants with little leaf symptoms, both collected in the mentioned peanut fields were also infected by similar strain of phytoplasma which proved to be identical with each of the molecular marker employed to the peanut-infecting agent in India. This is the first report on the association of ‘Ca. P. australasia’ (16SrII-D subgroup phytoplasma) with PnLL disease in India.

Cannabis sativa plants growing nearby sugarcane fields affected with grassy shoot disease at Muzaffarnagar, Uttar Pradesh, India, showed severe witches’ broom symptoms. The DNA from symptomatic Cannabis and sugarcane samples was isolated and subjected to nested PCR assays with universal phytoplasma-specific primer pairs (P1/P7 and R16F2n/R16R2). A DNA fragment of ~ 1.2 kbp was amplified in all the symptomatic samples of sugarcane and Cannabis with R16F2n/R16R2 primer pair but was absent in non-symptomatic samples. Pairwise sequence comparison, phylogeny and in silico RFLP analysis of 16S rDNA sequences confirmed the identity of a rice yellow dwarf phytoplasma group-related strain (16SrXI-B) associated with Cannabis which suggested its role as potential natural reservoir of SCGS phytoplasma in nature.

Val d’Agri is an important orchard area located in the Basilicata Region (Southern Italy). A phenomenon affecting cv. “Golden Delicious” apples which lead to tree death has been observed in the past several years in this area. This phenomenon has already been detected in about 20 hectares and is rapidly expanding. The symptoms observed were “scaly bark” and extensive cankers, mainly located in the lower part of the trunk, associated with wood decay. Dead plants ranged from 20% to 80% and, in many cases, trees were removed by farmers. In order to identify the causes of this phenomenon, investigations were started in autumn/winter 2019. In order to determine the possible causal agents, fungal and bacterial isolations, from symptomatic tissues, were performed in laboratory. Bacterial isolations gave negative results, whereas pure fungal cultures (PFCs) were obtained after 3–4 passages on potato dextrose agar (PDA) media. Genetic material was extracted from each PFC and amplified by PCR using three pairs of primers: ITS5/4, Bt2a/Bt2b and ACT-512F/ACT-783R. The amplicons were directly sequenced, and nucleotide sequences were compared with those already present in the NCBI GenBank nucleotide database. All isolated fungi were identified based on morphological features and multilocus molecular analyses. Neofusicoccum parvum, Diaporthe eres and Trametes versicolor were most frequently isolated, while Pestalotiopsis funerea, Phomopsis spp. and Diaporthe foeniculina were less frequently isolated. All nucleotide sequences obtained in this study have been deposited into the EMBL database. Pathogenicity tests showed that N. parvum was the most pathogenic and aggressive fungus, while Phomopsis sp. was demonstrated to be the less virulent one. All the investigated fungi were repeatedly reisolated from artificially inoculated twigs of 2-year-old apple trees, cv. “Golden Delicious”, and subsequently morphologically and molecularly identified. The role played by the above-mentioned fungi in the alterations observed in field is also discussed.

The incidence of sugarcane grassy shoot (SCGS) disease may reach up to 70% or more as is often true for ratoon crops resulting in yield losses of 40-90%, but often approaching 100% (Rishi and Chen, 1989; Viswanathan, 1997, 2001a; Rao and Dhumal, 2002; Srivastava et al., 2006).

RUS is of considerable economic importance, especially in areas where outbreaks occur. It has been described as a serious and widespread problem of raspberry in the former Soviet Union (see van der Meer (1987) for references). After the First World War, the disease became disastrous in raspberry areas in southern parts of The Netherlands, and many young plantations, started with healthy plant material, contained 60-90% infected plants in the second year after planting (see van der Meer (1987) for references). Small outbreaks and/or incidental occurrences of RUS disease have been reported from several other European countries (van der Meer, 1987).

Aster yellows (AY) group phytoplasmas affect plants by causing a general reduction in quantity and quality of yield. The most severely affected hosts are carrot, lettuce, onion, spinach and several ornamental crops, including aster, gladiolus, hydrangea, chrysanthemum and purple coneflower. Disease incidence may vary from year to year depending on the population trend of the vectors in the field. Infection rates varying from 20 to 30% were observed in lettuce and ranunculus fields in southern Italy (Marcone et al., 1995; Parrella et al., 2008). In Oklahoma, USA, according to Errampalli et al. (1991), AY group phytoplasma infections occurred in 80% of lettuce plants and 28% of carrots. In Ohio, disease incidence of 100% has been recorded in lettuce fields (Zhang et al., 2004). A major outbreak of AY disease occurred in 2000 in Texas that affected several vegetable crops. Among them, carrot was most severely damaged with infection rates that ranged from to 50 to near 100% (Lee et al., 2003). A severe AY disease of chrysanthemum which induced losses of 70 to 80% of the crop has been reported from China (Min et al., 2008) whereas losses of 90% were recorded in AY-affected aubergines in Bangladesh (Kelly et al., 2009). Infection rates of 60 and 99% were recorded in Hungary for AY-affected sugarbeet plants and India for AY-affected Jatropha curcas plants, respectively (Mumford et al., 2000; Kumar et al., 2010b). However, there are also several reports on sporadic occurrence and generally low incidence of AY group phytoplasmas in vegetable and ornamental crops (Smith et al., 1988; Vibio et al., 1995; Bertaccini et al., 1998). Cassava witches' broom, a disease affecting cassava in South East Asia (Vietnam, Thailand, Cambodia, Laos, China and the Philippines) is caused by 16SrI phytoplasma. The disease has resulted in significant reductions in cassava root starch content and up to 80% yield loss in parts of Vietnam (Anon., 2014).

This paper provides an updating of information of a selected number of major phytoplasma diseases of forest trees, with a focus on the associated phytoplasma taxa. Phytoplasma diseases of forest trees have been less extensively studied than those affecting fruit trees. Research on the role of phytoplasmas as the cause of diseases of forest trees has only in the last few years been intensified, after sensitive and specific detection methods greatly based on PCR technology became available. Various phytoplasma taxa have been identified in naturally infected elm, ash, conifer, sandal, and eucalyptus trees, whereas only one phytoplasma taxon has been recorded in naturally infected alder trees. However, for almost all of the reviewed diseases, there is still sparse information about insect vectors, plant host range, strain virulence, pathogenicity, and host tolerance and resistance. Knowledge of these aspects is the basis for appropriate disease management. In particular, further research is required to clarify the role of phytoplasmas in asymptomatic trees. In addition, the etiological role of various “non-specific” phytoplasma taxa, which have been recorded in forest trees, while no data from pathological studies are available, needs to be further investigated.

Symptoms typical of phytoplasma infection such as phyllody, virescence, witches’ broom and yellowing were observed in 12 varieties of Chrysanthemum morifolium in floral nurseries and experimental fields at New Delhi, Karnataka, Maharashtra and Andhra Pradesh, India, during surveys made from 2015 to 2017. Disease incidence ranged from 15 to 30%. Phytoplasma presence was confirmed in all symptomatic chrysanthemum varieties by molecular identification assays. Sequence comparison, phylogenetic and in silico RFLP analyses of 16S rDNA sequences allowed the identification of the chrysanthemum infecting phytoplasma strains into different ribosomal groups and subgroups, namely 16SrI, 16SrII-D, 16SrVI-D and 16SrXIV. Detection of phytoplasma strains of 16SrII-D subgroup were also confirmed in symptomatic Chenopodium album and Parthenium hysterophorus plants grown in and around the surveyed chrysanthemum fields at New Delhi, whereas 16SrVI-D phytoplasma strains were detected in symptomatic Cannabis sativa weed and leafhopper Hishimonus phycitis individuals collected from the symptomatic chrysanthemum fields at New Delhi. This is the first report on the presence of 16SrVI and 16SrXIV groups of phytoplasmas in chrysanthemum plants. Studies on genetic diversity of phytoplasmas infecting the major chrysanthemum varieties in India and their epidemiological aspects had previously not been reported. The detection and identification of phytoplasmas in different chrysanthemum varieties could contribute to increase the awareness among farmers in the management of these diseases.

Bermuda grass showing symptoms of a white leaf disease has been observed in fruit orchards, vegetable fields, and uncultivated areas in the Latium and Campania regions of central and southern Italy. Using polymerase chain reaction (PCR) amplification with phytoplasma-specific primers, all symptomatic plants tested positively; whereas no amplification product was obtained from nonsymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified ribosomal DNA revealed a uniform pattern that was similar to that of the Bermuda grass white leaf phytoplasma collected in Thailand, which is known to be a member of the sugarcane white leaf phytoplasma group. By RFLP analysis, the phytoplasma infecting Bermuda grass could be distinguished from other group members, including the phytoplasmas associated with sugarcane white leaf and Brachiaria white leaf. This is the first report on the presence of the Bermuda grass white leaf phytoplasma in Europe.

Phytoplasma diseases are difficult to control and one of the most promising approaches is through the use of resistant plants. Intra- and interspecific differences in the response of plants to various phytoplasma diseases have been observed over the last decades under both experimental and natural infection conditions. This chapter summarizes information on identification of resistant genotypes to a number of major phytoplasma diseases of temperate fruit trees, sesame, brinjal, coconut, jujube and forest trees and shrubs, for which the current knowledge is more advanced than that of other phytoplasma diseases. The resistant genotypes are suitable for disease management and should be regarded as a safe, effective and environmentally friendly control measure. Also, although some genotypes so far identified are not entirely satisfactory for agronomical purposes, they can be further exploited either in conventional breeding programs or through biotechnological approaches aimed at developing plants with suitable agronomic properties in which resistance to phytoplasma diseases is stably inserted.

This chapter presents five different procedures for extracting DNA from phytoplasma-infected plants and insect vectors suitable for PCR assays. One of these procedures enriches phytoplasma DNA through differential centrifugation and is effective in producing highly purified DNA from fresh tissues from a wide variety of herbaceous and woody plants. Although the DNA yield is less than those of other known total DNA extraction procedures, a major advantage of the presented phytoplasma-enriched procedure is that a substantial proportion of the isolated DNA is from phytoplasmas. The other four procedures here presented involve treatments with CTAB-based buffer to lyse cells and purify DNA followed by deproteination and recovery of DNA. These procedures work well for extracting total DNA from fresh, frozen, or lyophilized tissues from a wide variety of plant hosts as well as insect vectors. Because few manipulations are required, the CTAB-based procedures are faster and easier to perform than the phytoplasma-enrichment protocol. In addition, they result in very high yields and provide DNA that is less pure but of suitable quality for the use in standard molecular biological techniques including PCR assays.

This chapter presents five different procedures for extracting DNA from phytoplasma-infected plants and insect vectors suitable for PCR assays. One of these procedures enriches phytoplasma DNA through differential centrifugation and is effective in producing highly purified DNA from fresh tissues from a wide variety of herbaceous and woody plants. Although the DNA yield is less than those of other known total DNA extraction procedures, a major advantage of the presented phytoplasma-enriched procedure is that a substantial proportion of the isolated DNA is from phytoplasmas. The other four procedures here presented involve treatments with CTAB-based buffer to lyse cells and purify DNA followed by deproteination and recovery of DNA. These procedures work well for extracting total DNA from fresh, frozen, or lyophilized tissues from a wide variety of plant hosts as well as insect vectors. Because few manipulations are required, the CTAB-based procedures are faster and easier to perform than the phytoplasma-enrichment protocol. In addition, they result in very high yields and provide DNA that is less pure but of suitable quality for the use in standard molecular biological techniques including PCR assays.

Phytoplasma diseases of medicinal plants occur worldwide and are of great concern. So far 19 different phytoplasma ribosomal groups encompassing various subgroups have been reported. The subgroup 16SrI-B phytoplasmas are the prevalent agents mainly detected in Europe, North America and Asia. Phytoplasma diseases of medicinal plants severely reduce yield and quality of crops along with the longevity of the plants. Changes in the composition of secondary metabolites are induced, while the levels of valuable phytochemicals are greatly affected. In contrast, an accumulation of pharmaceutically important compounds such as vinblastine and vincristine is reported in periwinkle upon phytoplasma infections. Important phytoplasma diseases of several medicinal plants with special reference to their impact on active biological constituents and secondary metabolites are reviewed. General information on geographic distribution, diagnostics, genetic diversity, natural transmission and management aspects of phytoplasmas infecting medicinal plants are also discussed.

In the northern hemisphere, yellows, witches’ broom, and decline diseases of several forest and urban tree species are widespread and of considerable economic and ecological significance. Elm (Ulmus spp.) and alder (Alnus spp.) are affected by elm yellows (EY) and alder yellows (ALY), respectively. These diseases are mainly associated with the presence of closely related phytoplasmas, the EY agent ‘Candidatus Phytoplasma ulmi’ and the ALY agent, which are members of the EY or 16SrV group, subgroups 16SrV-A and 16SrV-C, respectively. Ash (Fraxinus spp.) is affected by ash yellows, a disease which occurs mainly in North America and is associated with the presence of ‘Candidatus Phytoplasma fraxini’, a member of subgroup 16SrVII-A. Poplar (Populus spp.), sandal (Santalum album), paulownia (Paulownia spp.), and mulberry (Morus spp.) are affected by yellows diseases associated with phytoplasmas of different 16SrI subgroups. Several species of conifers are affected by yellows and witches’ broom diseases associated with phytoplasmas belonging to at least five taxonomic groups (16SrI, 16SrIII, 16SrVI, 16SrIX, and 16SrXXI) and several different subgroups. A number of urban tree species grown in the Sabana de Bogota (Colombia) are affected by decline diseases which are primarily associated with 16SrI and 16SrVII phytoplasmas. This chapter summarizes the current knowledge of major phytoplasma diseases of forest and urban trees grown in the northern hemisphere.

Peach (Prunus persica) is in many countries severely affected by yellows and decline diseases of phytoplasmal etiology. These diseases are caused by phylogenetically different phytoplasmas. X-disease is one of the most serious diseases of peach. It is widespread in North America and has not been observed elsewhere. This disease is induced by a distinct phytoplasma, the X-disease agent ‘Candidatus Phytoplasma pruni’, which is a member of the X-disease phytoplasma group or 16SrIII group, subgroup 16SrIII-A. ‘Ca. P. pruni’ is, at the level of 16S rDNA sequences, a homogeneous pathogen. Peach rosette, peach red suture, and peach yellows, also called little peach, are three other diseases which are mainly known from eastern North America. These diseases are currently of minor importance. They are also caused by the X-disease phytoplasma. Peach yellow leaf roll (PYLR) is a major disease of peach that occurs in California. The causative agent, the PYLR phytoplasma, is a member of the apple proliferation (AP) group and is most closely related to the pear decline phytoplasma. In Europe, peach is affected by an economically important disorder known as European stone fruit yellows. This disorder is induced by ‘Ca. P. prunorum’ which is a member of the AP group, subgroup 16SrX-B. ‘Ca. P. prunorum’ is closely related to other important temperate fruit tree phytoplasmas including the PYLR agent. In Lebanon and Iran, peach is affected by almond witches’-broom, a lethal disease that is induced by ‘Ca. P. phoenicium’. This phytoplasma is a member of the 16SrIX group, subgroup 16SrIX-B. Phytoplasmas of other phylogenetic groups, which are known to infect a wide range of plant hosts, have been identified in declining peach trees in several fruit growing areas worldwide. However, the pathological relevance of these non-peach phytoplasmas needs to be investigated in many cases.

Aim: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Materials and Results: Phytoplasma isolates belonging to stolbur subgroup 16SrXII-A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf-a type and 68% by tuf-b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf-b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences’ analysis and virtual digestions. Conclusions: The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Significance and Impact of the Study: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.

In several sugarcane-growing areas of India, sugarcane plants showing typical symptoms of sugarcane grassy shoot (SCGS) disease as well as non-symptomatic sugarcane plants and sugarcane with non-specific yellowing and stunting symptoms were examined for phytoplasmal infections by means of polymerase chain reaction (PCR) technology, using primers directed to phytoplasma ribosomal DNA (rDNA) sequences. From all field-collected, symptomatic sugarcane plants and those with non-specific symptoms as well as from most the non-symptomatic plants, detectable PCR products were obtained through the highly sensitive nested-PCR assays. Also, the phylogenetic relationships of SCGS phytoplasma strains including those whose rDNA sequences were determined in this study, to each other and to other closely related phytoplasmas, at both 16S rRNA gene and 16S/23S rDNA spacer region sequence levels were examined. The SCGS phytoplasma is a member of the rice yellow dwarf (RYD) phytoplasma group or 16SrXI group, and proved very closely related to the sugarcane with leaf (SCWL) agent. More distantly related to the SCGS agent, are sorghum grassy shoot (SGS), Bermuda grass white leaf (BGWL) (=‘Candidatus Phytoplasma cynodontis’) and RYD (= ‘Candidatus Phytoplasma oryzae’) phytoplasmas. The extensive survey of phytoplasmal infections in sugarcane plants in India as well as analysis of phylogenetic relationships of SCGS phytoplasma strains to each other and to other closely related phytoplasmas, at both 16S rRNA gene and 16S/23S rDNA spacer region sequence levels, had previously not been known.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon ‘Candidatus Phytoplasma asteris’. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in Candidatus’. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S–23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name ‘Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S–23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.

Sugarcane is severely affected by yellows and decline diseases of phytoplasmal etiology. These diseases which are of considerable economic importance, cause similar symptoms but differ in the identity of the associated phytoplasmas, vectorship and geographic distribution. Sugarcane white leaf (SCWL) and sugarcane grassy shoot (SCGS) occur only in the south-east Asian region and not in other sugarcane growing areas of the world. Both are caused by a single phytoplasma type that is a member of the SCWL group and appears to infect only sugarcane. However, SCWL and SCGS phytoplasmas are closely related organisms and could be differentiated by RFLP analysis of rDNA using suitable restriction endonucleases. Sugarcane yellow leaf syndrome (SCYLS) disease has been reported from several African countries, Cuba and Australia, and is associated with distinctly different phytoplasmas which are not specific pathogens. They include members of the X-disease, faba bean phyllody, aster yellows, SCWL, LfWB and CirP groups which are known to infect a wide range of wild and cultivated plants and have a low insect vector specificity. Ramu stunt disease of sugarcane (SCRS) is known to occur in Papua New Guinea. This disease is associated with a SCWL-related phytoplasma and is presumed to be naturally spread by the leafhopper Eumetopina flavipes. Sugarcane green grassy shoot (SCGGS) is a newly discovered phytoplasmal disease of sugarcane in Thailand.

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.

A physical map of the European stone fruit yellows phytoplasma strain GSFY1 chromosome was constructed using PFGE-purified genomic DNA from diseased tobacco and tomato plants. The map was generated with single and double digestions of the chromosome with SmaI, BssHII, ApaI, BamHI and XhoI restriction endonucleases and the fragments were resolved by PFGE. Reciprocal double digestions were used to locate 26 restriction sites on the chromosome. Southern blot analysis was also used to assist in the arrangement of the contiguous restriction fragments obtained. From the restriction fragments generated by double digestion, the circular chromosome was calculated to be approximately 635 kb. Loci of two rRNA operons, the operon containing the tuf gene, genes encoding an immunodominant membrane protein and a putative nitroreductase, and randomly cloned DNA fragments IH184 and AT67 were placed on the map. Digestion of chromosomal DNA of strain GSFY1 with MluI gave a complex restriction pattern, suggesting that this isolate consists of a population with heterogeneity with respect to MluI restriction sites. The GSFY1 physical map was different from that of the closely related apple proliferation phytoplasma but the genetic arrangement was similar.

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene. On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16SrI-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three new subgroups that differ in the RFLP profiles were identified and designated 16SrI-L, 16SrI-M and 16SrI-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 bp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16SrI-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16SrI-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16SrI-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16SrI-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.

An immunodominant membrane protein (IMP) of the apple proliferation (AP) phytoplasma was detected in preparations from AP-diseased periwinkle plants using monoclonal and polyclonal antibodies to the AP agent. Following isolation from Western blots and partial sequencing, degenerate oligonucleotides derived from the IMP sequence were used as probes to identify a DNA fragment containing the ORF encoding the IMP. Complete sequencing and subsequent analysis of the cloned DNA fragment revealed the presence of two ORFs, predicted to encode proteins with molecular masses of 25 kDa (P-318A) and 19 kDa (P-318B). Whilst database searches failed to assign a possible function to P-318A, analysis of P-318B predicted an amphiphilic membrane protein with a positively charged N-terminal portion, followed by a hydrophobic segment forming an alpha-helix, and a hydrophilic C-terminal part located outside of the cell. The amphiphilic nature of P-318B was confirmed by its solubility in Triton X-114. The gene encoding P-318B was expressed in Escherichia coli and the resulting protein was used to immunize rabbits. The antiserum obtained reacted specifically with P-318B. The same protein was also detected by an antiserum raised against antigen preparations from AP-diseased plants. The P-318B antiserum did not react with antigen preparations from plants infected with the closely related pear decline phytoplasma. However, in Southern hybridization studies, the gene encoding the IMP hybridized to genomic fragments of the pear decline and European stone fruit yellows phytoplasmas. It also showed significant sequence similarity to a gene encoding an antigenic membrane protein of the sweet potato witches' broom phytoplasma, but not to a gene encoding a major immunogenic membrane protein of an aster yellows group phytoplasma. Since it appears that most phytoplasmas possess a major immunogenic membrane protein which may have a function in pathogenesis, this work may be a basis for further studies on fundamental aspects of host-pathogen interactions. It also describes a new approach to obtain suitable immunogens to produce specific antibodies for detection and characterization of the non-culturable phytoplasmas.

Summary The alder yellows phytoplasma (ALY) was transmitted to the experimental host Catharanthus roseus (periwinkle) via dodder (Cuscuta odorata) bridges from naturally infected alder trees. The identity of the dodder-transmitted phytoplasma has been confirmed by restriction fragment length polymorphism (RFLP) analyses of polymerase chain reaction (PCR)-amplified ribosomal DNA.

Restriction fragment length polymorphism and sequence analysis of PCR-amplified ribosomal DNA were used to identify and classify phytoplasmas associated with diseases of various wild and cultivated plants. The diseases examined were either not known before or the presumable causal agents were not yet identified and characterized or were only known from other geographic areas. New diseases examined were those causing virescence and phyllody of Bunias orientalis and Cardaria draba. Both were associated with strains of the aster yellows phytoplasma. The same type of aster yellows phytoplasma was also found to be associated with yellows and phyllody diseases of Portulaca oleracea, Stellaria media, Daucus carota ssp. sativus, and Cyclamen persicum. In German and French DNA samples from diseased Trifolium repens, the clover phyllody phytoplasma was identified, which could clearly be distinguished from other phytoplasmas of the aster yellows group. Strains of the stolbur phytoplasma were detected in big bud-affected tomatoes and almost exclusively in Convolvulus arvensis. In Cirsium arvense and Picris echioides two distinct phytoplasmas were identified which showed relationship to the sugarcane white leaf phytoplasma group but may represent a new group or subgroup. In Conyza (syn.: Erigeron) canadensis a phytoplasma of the X-disease group was detected. A strain from Gossypium hirsutum showed the same restriction profiles as the faba bean phyllody phytoplasma.

In the Campania region of southern Italy, commercial orchards of European hazel (Corylus avellana) are severely affected by yellowing and decline. To determine whether phytoplasmas are associated with the disorder, stem samples from diseased trees were examined using polymerase chain reaction assays. No visible products were obtained by amplification of sample DNA with universal and group-specific phytoplasma primers. However, when the products obtained with universal primers were re-amplified with nested primers that were specific for the fruit tree phytoplasmas of the apple proliferation group, most samples tested positively. Restriction site analysis revealed that the trees were infected with the apple proliferation, pear decline, and European stone fruit yellows phytoplasmas in about the same proportion. Some of the trees were doubly infected with one of the fruit tree phytoplasmas and the aster yellows agent. Most of the infected trees were also identified by hybridization of the products obtained in the initial amplification with suitable oligonucleotide probes.

Summary Elm yellows (EY) is a lethal or decline phytoplasma disease that affects several Ulmus (elm) species and hybrids, which is widespread in North America and Europe. The symptoms vary among the elm species. In those native to North America, main symptoms include epinasty, chlorosis, premature casting of the leaves, yellow to brown discoloration of the phloem in the roots and stem and tree death that usually occurs within 1 or 2 years from the appearance of foliar symptoms. In contrast, affected trees of European and Asian species are primarily characterized by witches’ broom as a specific symptom, do not show phloem discoloration and are less prone to decline. The disease is caused by a relatively genetically homogeneous phytoplasma, the EY agent “Candidatus Phytoplasma ulmi,” a member of the EY phytoplasma group or 16SrV group, subgroup 16SrV-A. In nature, this pathogen exhibits a high plant host specificity. The elm leafhopper Scaphoideus luteolus is the only confirmed vector of EY phytoplasma in North America, whereas Macropsis mendax has been reported as a natural vector in Northern Italy. However, other insect vectors are likely to be involved in its natural spread. Phytoplasmas of other taxonomic groups or 16SrV subgroups, which are known to infect a wide range of plant hosts, have been identified in naturally infected elm trees. However, the pathological relevance of these “non-elm” phytoplasmas needs to be confirmed in many cases. Their detection is mainly based on the highly sensitive nested PCR assays, while pathological data are lacking. This study summarizes, within the framework of a single comprehensive review, the current knowledge of EY. Gaps in knowledge of this disease and prospects for future research are also critically discussed.