Yellow leaf syndrome modifies the composition of sugarcane juices in polysaccharides, phenols and polyamines

Some ultrastructural changes can be observed in diseased Saccharum officinarum L. (cv. Cuba 120-78) plants with visual symptoms of yellow leaf syndrome (YLS), used to discriminate between healthy and diseased plants. Abaxial epidermis of diseased leaves shows a large amount of adhered superficial bodies, which partially occluded some stomata. Bundle sheath cells surrounding the bottom of phloem of diseased leaves are separated from the conducting tissues by a large layer of an amorphous matrix similar to wax. Debris of the end wall can be observed in large xylem vessels. Sometimes, spherical bodies similar to phytoplasma can be observed in the intercellular spaces of bundle sheath cells. These particles have never been observed in healthy plants. YLS was also associated to an increase of the concentration of reducing sugars, glucose index, and glycoproteins recovered in juices whereas the amount of sucrose decreases. Sugarcane juices obtained from both healthy and YLS-affected Cuba 120-78 cultivars of sugarcane contained putrescine (PUT), cadaverine (CAD), spermidine and spermine (SPM) as free and macromolecules-conjugated compounds. Only CAD and SPM appeared as acid-soluble conjugates to small molecules whereas PUT and CAD are the major polyamines (PAs) conjugated to macromolecules, mainly to high molecular mass glycoproteins. The disease was associated to an increase in total PA fraction. Arginase and ornithine decarboxylase activities, responsible for the synthesis of PUT, were higher in YLS juices than in those obtained from healthy plants. CAD and SPM presumably conjugated mostly to chlorogenic, syringic and ferulic acids in juices from YLS plants.     

Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes

Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma strain Aster Yellows-Witches' Broom (AY-WB) and Spiroplasma kunkelii are near completion. Complete genome sequences of seven obligate animal and human pathogenic mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii were compared to those of the completed genomes. This resulted in identification of at least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts. Phylogenetic analyses using parsimony were employed to study the origin of these genes, resulting in identification of one gene that may have undergone horizontal gene transfer. The possible involvement of these genes in plant pathogenicity is discussed.     

Molecular study of phytoplasmas associated with pistachio yellows in Iran

A large‐scale survey was conducted on pistachio plants exhibiting foliar symptoms including scorch, little leaf, yellows and reddish in pistachio growing areas in the Qom, Yazd and Qazvin provinces of Iran. Total DNA was extracted from symptomatic and symptomless pistachio and used in nested PCR assays with phytoplasma universal primers. Nested PCR products were obtained for symptomatic plant samples while the symptomless plants yielded no PCR products. Virtual restriction fragment length polymorphism, phylogenetic and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to two ribosomal groups; in particular, “Candidatus Phytoplasma solani” and “Ca. P. phoenicium” were identified. The presence of these phytoplasmas in pistachio is of great phytosanitary significance due to its commercial interest.     

Multigene Sequence Analysis of Aster Yellows Phytoplasma Associated with Primrose Yellows

Primula acaulis (L.) Hill. plants showing stunting, leaf‐yellowing and virescence were first discovered in the Czech Republic. Polymerase chain reactions with subsequent restriction fragment length polymorphism analyses and sequencing enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI‐B, tufI‐B, rpI‐B, groELIB‐III and SecY‐IB subgroups. Phylogeny of the 16S rRNA gene sequences as well as sequence analysis of several chromosomal regions, such as the 16S‐23S ribosomal operon, ribosomal proteins, spc ribosomal protein operon, genes for elongation factor EF‐Tu, molecular chaperonin large subunit GroEL, immunodominant membrane protein, ribosome recycling factor, urydilate kinase, ATP‐ and Zn²⁺‐dependent proteases not only confirmed its affiliation with the ‘Candidatus Phytoplasma asteris’ species but also enabled its detailed molecular characterization. The less researched regions of phytoplasma genome (amp, adk, hflB, pyrH‐frr genes) could be valuable as additional markers for phytoplasma through differentiation especially within the 16SrI‐B ribosomal subgroup.     

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.     

Comparative proteomic analysis provides new insights into mulberry dwarf responses in mulberry (Morus alba L.)

Mulberry dwarf (MD) is a serious infectious disease of mulberry caused by phytoplasma. Infection with MD phytoplasma results in stress phenotypes of yellowing, phyllody, stunting, proliferation, and witches' broom. Physiological and biochemical analysis has shown that infection with MD phytoplasma causes an increase in soluble carbohydrate and starch content, and a decrease in the net photosynthesis rate, carboxylation efficiency, and pigment content of leaves. Furthermore, damage to the chloroplast ultrastructure was detected in infected leaves. To better understand the pathogen‐stress response of mulberry (Morus alba L.) to MD phytoplasma, we conducted a comparative proteomic analysis using 2‐DE of infected and healthy leaves. Among 500 protein spots that were reproducibly detected, 20 were down‐regulated and 17 were up‐regulated. MS identified 16 differentially expressed proteins. The photosynthetic proteins rubisco large subunit, rubisco activase, and sedoheptulose‐1,7‐bisphosphatase showed enhanced degradation in infected leaves. Based these results, a model for the occurrence mechanism of MD is proposed. In conclusion, this study provides new insights into the mulberry response to MD phytoplasma infection.     

Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragariaxananassa Duch.) using PCR

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.     

16S rRNA interoperon sequence heterogeneity distinguishes strain populations of palm lethal yellowing phytoplasma in the Caribbean region

DNA of phytoplasmas in lethal yellowing (LY)‐diseased palms was detected by a nested polymerase chain reaction (PCR) assay employing rRNA primer pair P1/P7 followed by primer pair LY16Sf/ LY16‐23Sr. Polymorphisms revealed by Hinfl endonuclease digestion of rDNA products differentiated coconut‐infecting phytoplasmas in Jamaica from those detected in palms in Florida, Honduras and Mexico. A three fragment profile was generated for rDNA from phytoplasmas infecting all 21 Jamaican palms whereas a five fragment profile was evident for phytoplasmas infecting the majority of Florida (20 of 21), Honduran (13 of 14) and Mexican (5 of 5) palms. The RFLP profile indicative of Florida LY phytoplasma was resolved by cloning into two patterns, one of three bands and the other of four bands, that together constituted the five fragment profile. The two patterns were attributed to presence of two sequence heterogeneous rRNA operons, rrnA and rrnB, in most phytoplasmas composing Florida, Honduran and Mexican LY strain populations. Unique three and four fragment RFLP profiles indicative of LY phytoplasmas infecting Howea forsteriana and coconut palm in Florida and Honduras, respectively, were also observed. By comparison, the Jamaican LY phytoplasma population uniformly contained one or possibly two identical rRNA operons. No correlation between rRNA interoperon heterogeneity and strain variation in virulence of the LY agent was evident from this study.     

First Report of Phytoplasma (16SrI) Associated with Yellow Decline Disease of Royal Palms [Roystonea regia (Kunth) O. F. Cook] in Malaysia

Royal Palms (Roystonea regia) with symptoms such as severe chlorosis, stunting, collapse of older fronds and general decline were observed in the state of Selangor, Malaysia. Using polymerase chain reaction (PCR) amplification with phytoplasma universal primer pair P1/P7 followed by R16F2N/R16R2 and fU5/rU3 as nested PCR primer pairs, all symptomatic plants tested positively for phytoplasma. Results of phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences revealed that the phytoplasma associated with Royal Palm yellow decline (RYD) was an isolate of ‘Candidatus Phytoplasma asteris’ belonging to a new 16SrI‐subgroup. These results show that Roystonea regia is a new host for the aster yellows phytoplasma (16SrI). This is the first report on the presence of 16SrI phytoplasma on Royal Palm trees in Malaysia.