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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.

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.

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 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.

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.

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.

Pulsed-field gel electrophoresis (PFGE) is a powerful technique for genomic studies of unculturable plant-pathogenic phytoplasmas, which enables separation of full-length phytoplasma chromosomes from contaminating host plant nucleic acids. The PFGE method described here involves isolation of phytoplasmal DNA from high-titer phytoplasma-infected herbaceous plants using a phytoplasma enrichment procedure, embedding of phytoplasma chromosomes in agarose blocks, and separation of entire phytoplasma chromosomes from contaminating host plant nucleic acids by electrophoresis. Full-length phytoplasma chromosomes are resolved as single, discrete bands in the gel. The identity of these bands can be confirmed by Southern blot hybridization using a ribosomal DNA fragment as a probe. The method does not utilize gamma-irradiation to linearize phytoplasma chromosomes prior to electrophoresis.

In southern Italy, Spartium junceum (Spanish broom) is severely affected by a phytoplasmal disease, Spartium witches’‐broom (SpaWB). The volatile fractions extracted from flowers of healthy and diseased plants, examined by gas chromatography and gas chromatography–mass spectrometry, appeared to be quantitatively and qualitatively different. In both the healthy and the diseased plants, the main components were n‐alkanes, which occurred at a rate of 55.2% and 38.8%, respectively. The level of aliphatic acids was considerably lower in flowers of the diseased plants than in those of the healthy plants (4.5% vs. 18.7%). Sesquiterpenes were detected only in the diseased plants. It is possible that the changes in the composition of secondary metabolites of diseased plants can be related to plant defense responses. Abbreviations: AP, apple proliferation; EY, elm yellows; SpaWB, Spartium witches’‐broom

A phytoplasma has been detected in Oplismenus burmannii (Retz.) P. Beauv. and Digitaria sanguinalis (L.) Scop. plants with symptoms of chlorotic streaks and white leaf disease in India. By sequence and phylogenetic analyses of polymerase chain reaction-amplified rDNA sequences, the detected phytoplasma was identified as ‘Candidatus Phytoplasma cynodontis’, a member of the 16SrXIV group, and the causal agent of Bermuda grass white leaf disease. This is the first report on the occurrence of ‘Ca. Phytoplasma cynodontis’ in O. burmannii and D. sanguinalis grasses.

Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.

The trivial name ‘phytoplasma’ has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus ‘Candidatus (Ca.) Phytoplasma’. Given the diversity within ‘Ca. Phytoplasma’, several subtaxa are needed to accommodate organisms that share Ca. Phytoplasma’, a taxon that includes the species ‘Ca. Phytoplasma aurantifolia’ (the prokaryote associated with witches'-broom disease of small-fruited acid lime), ‘Ca. Phytoplasma australiense’ (associated with Australian grapevine yellows), ‘Ca. Phytoplasma fraxini’ (associated with ash yellows), ‘Ca. Phytoplasma japonicum’ (associated with Japanese hydrangea phyllody), ‘Ca. Phytoplasma brasiliense’ (associated with hibiscus witches'-broom in Brazil), ‘Ca. Phytoplasma castaneae’ (associated with chestnut witches'-broom in Korea), ‘Ca. Phytoplasma asteris' (associated with aster yellows), ‘Ca. Phytoplasma mali’ (associated with apple proliferation), ‘Ca. Phytoplasma phoenicium’ (associated with almond lethal disease), ‘Ca. Phytoplasma trifolii’ (associated with clover proliferation), ‘Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), ‘Ca. Phytoplasma ziziphi’ (associated with jujube witches'-broom), ‘Ca. Phytoplasma oryzae’ (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence dorée, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97·5 % similar to those of other ‘Ca. Phytoplasma’ species, are characterized by distinctive biological, phytopathological and genetic properties. These include ‘Ca. Phytoplasma pyri’ (associated with pear decline), ‘Ca. Phytoplasma prunorum’ (associated with European stone fruit yellows), ‘Ca. Phytoplasma spartii’ (associated with spartium witches'-broom), ‘Ca. Phytoplasma rhamni’ (associated with buckthorn witches'-broom), ‘Ca. Phytoplasma allocasuarinae’ (associated with allocasuarina yellows), ‘Ca. Phytoplasma ulmi’ (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being Ca. Phytoplasma’ species, are not presently described as Candidatus species, due to their poor overall characterization.

Yellows-diseased plants of Crepis setosa (hawksbeard), Knautia arvensis (field scabious), Convolvulus arvensis (field bindweed), Picris echioides (bristly oxtongue), Echium vulgate (blueweed) and Calendula officinalis (pot marigold) collected in central and southern Italy were examined for phytoplasma infection by means of polymerase chain reaction (PCR) technology using universal phytoplasma primers directed to ribosomal sequences. The detected phytoplasmas were characterized and differentiated using restriction fragment length polymorphism analysis of PCR-amplified DNA. The phytoplasma detected in diseased pot marigold plants was identified as a member of the aster yellows group and proved indistinguishable from a strain of the American aster yellows phytoplasma. The phytoplasma identified in diseased field bindweed plants is a putative new type of the stolbur group that differed from the typical stolbur phytoplasma. Phytoplasmas detected in diseased hawksbeard, blueweed and field scabious plants are all putative new members of the sugarcane white leaf group while the phytoplasma detected in diseased bristly oxtongue plants represents a new member of the faba bean phyllody group. For hawksbeard and field scabious this is the first report on the occurrence of phytoplasma diseases, whereas phytoplasmas infecting bristly oxtongue and blueweed have never been characterized before.

Plant-pathogenic phytoplasmas are wall-less, unculturable bacteria of the class Mollicutes that are 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 reside in the phloem sieve tubes and are transmitted from plant to plant by phloem-feeding homopteran insects, mainly leafhoppers (Cicadellidae) and planthoppers (Fulgoromorpha), and less frequently psyllids (Psyllidae). Most of the host plants of phytoplasmas are angiosperms in which a wide range of specific and non-specific symptoms are induced. Phytoplasmas, which are phylogenetically more closely related to acholeplasmas than to other mollicutes, have a complex life cycle that involves colonization of different environments, the plant phloem and various organs of the insect vectors. The dynamic architecture of phytoplasma genomes may account for variation in their genome size and adaptation to the diverse environments of their plant and insect hosts. Newly acquired knowledge has made it possible to identify a considerable number of phytoplasma 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.

During the late summer of 2003, a wilt disease of the weed Italian cockleburr (Xanthium italicum Mor.) was observed in the Basilicata Region of southern Italy. Diseased plants were growing near an apricot orchard in which some trees were severely affected by Verticillium wilt. The most characteristic symptoms of the wilt disease affecting Italian cockleburr were yellowing, stunting, and gradual wilting. Also, diagonal and cross sections of stems revealed brown discoloration of their vascular tissues. To elucidate the etiology of the disease, we attempted detection and identification of the causal agents using traditional and polymerase chain reaction (PCR)-based methods. Small pieces of petiole and stem tissues from diseased and asymptomatic plants were surface disinfested in NaOCl solution, rinsed in sterile distilled water, blotted dry, and plated onto water agar (WA) medium. Following incubation at 22°C, the emerging colonies were transferred to potato dextrose agar (PDA). Verticillium dahliae (one isolate) was consistently identified on the basis of its morphological features according to the description of Smith (2). Using PCR assays with the primer pair ITS5/ITS4 (3), which are directed to fungal nuclear ribosomal DNA (rDNA) repeat sequences, an amplification product of approximately 560 bp was obtained by using total DNA extracted from wilt-affected Italian cockleburr plant tissues (five plants examined) as well as fresh mycelium from the V. dahliae-infected Italian cockleburr pure culture-maintained isolate mentioned above. No visible PCR products were obtained with total DNA from asymptomatic Italian cockleburr plants. Sequence analysis of the ITS5/ITS4 amplicons revealed no differences in their nucleotide positions. The obtained sequence of the V. dahliae-infected Italian cockleburr isolate (GenBank Accession No. AJ865691) was then used as query sequences in a BLAST 2.0 search (1). Sequence of the southern Italian isolate proved to be identical to that of the Greek strain “76 Greece” of V. dahliae (GenBank Accession no. AF104926). To prove Koch's postulates, 10 healthy Italian cockleburr seedlings were experimentally inoculated by dipping trimmed roots in a single-conidial suspension (1.5 × 106 CFU/ml) obtained from 10-day-old colonies of the V. dahliae-infected Italian cockleburr pure culture-maintained isolate. After 4 weeks, all inoculated Italian cockleburr plants showed symptoms identical to those of naturally infected field-grown plants. V. dahliae was consistently reisolated from inoculated plants. Additional inoculation experiments revealed that pepper and eggplant were also susceptible to the V. dahliae-infected Italian cockleburr isolate showing typical Verticillium wilt symptoms. To our knowledge, this is the first report of the occurrence of Verticillium wilt of X. italicum. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. C. Smith. N.Z. J. Agric. Res. 8:450, 1965. (3) T. J. White et al. Pages 315–322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.