cDNA-AFLP analysis of gene expression changes in apple trees induced by phytoplasma infection during compatible interaction

peer reviewedAbstract In order to gain insight into molecular and physiological changes in apple trees during compatible interaction with two ‘Candidatus Phytoplasma mali’ strains (AP and AT), cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was used. A rootstock of apple (MM106) susceptible to ‘Ca. P. mali’ was used to extend the range of the potential host responses by the maximum number of identified genes that will be deregulated by phytoplasma in apple. Gene expression comparisons were studied in three directions: healthy versus infected samples, symptomatic versus nonsymptomatic sample, and AP-infected versus ATinfected sample. Forty-five genes whose steady-state levels of expression significantly changed in response to phytoplasma infection were identified. Among their partial cDNA sequences, only 27 showed similarity to DNA or protein data bases; of these, 18 were related to known genes in plants, and the rest were related to unknown or hypothetical proteins. Eighteen out of 45 did not show any similarity with sequences in data bases (potential novel genes). Quantitative real-time RT-PCR (qRT-PCR) was used to confirm differential expression of AFLP identified genes, and showed the similar profile expression for 11 known genes among 18, and for 13 unknown, hypothetical or novel genes among 27. Changes in gene expression involved a wide spectrum of biological functions, including processes of metabolism, cell defence, senescence, photosynthesis, transport, transcription, signal transduction and protein synthesis. This is the first study of global gene profiling in plants in response to phytoplasma infections using cDNA-AFLP, and a model is proposed to explain the mode of action of the ‘Ca. P. mali’ in apple

Genetic diversity of 'Candidatus Phytoplasma mali' strains in Poland

During 2010-2013, samples from 267 apple trees growing in six regions of Poland were tested for phy- toplasma presence. ‘Candidatus Phytoplasma mali’ was detected in 17 samples from phloem tissue of apple shoots showing typical apple proliferation symptoms. Molecular characterization of these strains was conducted using PCR-RFLP and sequence analyses of three regions: ribosomal DNA, ribosomal protein and non-ribosomal DNA fragment including nitroreductase and rhodanese-like protein genes. Fragments of 16S rDNA plus 16S-23S spacer region from the ‘Ca. P. mali’ strains showed two restriction profiles: P-I and P-II when digested with HpaII enzyme. Moreover, based on results of nitroreductase and rhodanese-like protein genes, these phytoplasmas were grouped into two subtypes: AP-15 and AT-1. The majority of the ‘Ca. P. mali’ strains was classified to the AP-15 subtype. Two strains were grouped to the AT-1 subtype and another showed both AP-15 and AT-1 profiles. Based on results of analysis of rpl22 and rps3 ribosomal protein genes, most of the ‘Ca. P. mali’ strains were identified as belonging to rpX-A. Only one strain was affiliated to rpX-B subgroup

Spiroplasma spp.: A Plant, Arthropod, Animal and Human Pathogen

Mollicutes is a class of smallest and free-living bacteria. They have no cell wall and their plasma membrane contains cholesterol; nevertheless, cellular organization does not differ from that of other prokaryotes. They are used as simple model systems for studying general biological problems, such as those concerning membrane structure and functions, symbiosis between arthropods and microrganisms, animal and plant pathogens. Mollicutes includes the family of Spiroplamataceae, which contains Spiroplasma genus, a group of species associated, in different manner, with arthropods (insects, mites, crustaceans). Spiroplasma species can be commensals or parasites and even be involved in more close symbiosis, such as synergism or mutualism. Out of 38 described Spiroplasma species, only three have been associated with plant diseases and three with arthropod diseases. Moreover, some species have been related to animal diseases, such as transmissible spongiform encephalopathy (TSE), and their role in human disease has been assessed. The chapter describes the taxonomic situation of the genus and reports the most important diseases due to the presence of Spiroplasma in different living organisms with special emphasis on citrus in which it causes one of the most economically damaging infectious diseases in a number of citrus growing areas worldwide

Identification and GroEL gene characterization of green petal phytoplasma infecting strawberry in Italy

none5The presence of phytoplasmas in strawberry showing malformation of the fruits together with the typical green petals symptoms was detected in some North Western Italy cultivations. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rDNA and GroEL sequences specific for phytoplasmas. Bands of 1.2 kb were obtained in both cases after nested-PCR assays and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows subgroup 16SrI-C, the GroELI grouping confirm all the strains from strawberry to be identical to each other and to GroELI-VI group. This is the first multigene molecular identification of strawberry green petals phytoplasmas in Italy.openContaldo N.; J.F. Mejia; S. Paltrinieri; A. Calari; A. BertacciniContaldo N.; J.F. Mejia; S. Paltrinieri; A. Calari; A. Bertaccin

Molecular identification and seasonal monitoring of phytoplasmas infecting Croatian grapevines

Phytoplasmas of the 16S rRNA RFLP group XII-A (stolbur) have been detected with tests carried out 4 times from September 1997 to April 1998 in Croatian Pinot gris grapevines showing yellows symptoms. This is the first report on the presence of stolbur phytoplasma in Croatian grapevines. Two asymptomatic vineyard weeds, Taraxacum officinale Web. and Polygonum lapathifolium L. showed the presence of the same prokaryotes

Potential Applications and Limitations of Electronic Nose Devices for Plant Disease Diagnosis

Electronic nose technology has recently been applied to the detection of several plant diseases and pests, with promising results. However, in spite of its numerous advantages, including operational simplicity, non-destructivity, and bulk sampling, drawbacks include a low sensitivity and specificity in comparison with microbiological and molecular methods. A critical review of the use of an electronic nose for plant disease diagnosis and pest detection is presented, describing the instrumental and procedural advances of sensorial analysis, for the improvement of discrimination between healthy and infected or infested plants. In conclusion, the use of electronic nose technology is suggested to assist, direct, and optimise traditionally adopted diagnostic technique

Pantoea agglomerans-Induced Dieback in Pistachio in Chile

Pistachio crops have a great economic potential, as their global production has increased dramatically over the past few decades. Therefore, it is important to maintain the healthy phytosanitary status of pistachio crops. In a Chilean pistachio orchard, a dieback of the trees was observed, with blighting of twigs and severe necrosis in the trunk and twigs. Bacterial isolation, pathogenicity tests and molecular characterization were conducted to determine the causal agent of the observed disease. The bacterial isolation and analysis of 16S rRNA gene led to the identification of Pantoea genus bacteria. Pathogenicity tests carried out on fruits inoculated with Pantoea isolates induced large necrosis seven days post-inoculation. Further inoculations were carried out on pruning cuttings and on the trunk of 18-month-old pistachio plants. Thirty-one weeks after inoculation, necrotic lesions were observed in the wood of pistachio plants. Sequence analysis of housekeeping genes enabled the isolated bacterium to be identified as Pantoa agglomerans, and to verify its role as the causal agent of the pistachio dieback with necrotic lesions. This is the first report of an occurrence of P. agglomerans inducing dieback in pistachio

Genetic diversity of ‘Candidatus Phytoplasma mali’ strains in Poland

During 2010-2013, samples from 267 apple trees growing in six regions of Poland were tested for phytoplasma presence. ‘Candidatus Phytoplasma mali’ was detected in 17 samples from phloem tissue of apple shoots showing typical apple proliferation symptoms. Molecular characterization of these strains was conducted using PCR-RFLP and sequence analyses of three regions: ribosomal DNA, ribosomal protein and non-ribosomal DNA fragment including nitroreductase and rhodanese-like protein genes. Fragments of 16S rDNA plus 16S-23S spacer region from the ‘Ca. P. mali’ strains showed two restriction profiles: P-I and P-II when digested with HpaII enzyme. Moreover, based on results of nitroreductase and rhodanese-like protein genes, these phytoplasmas were grouped into two subtypes: AP-15 and AT-1. The majority of the ‘Ca. P. mali’ strains was classified to the AP-15 subtype. Two strains were grouped to the AT-1 subtype and another showed both AP-15 and AT-1 profiles. Based on results of analysis of rpl22 and rps3 ribosomal protein genes, most of the ‘Ca. P. mali’ strains were identified as belonging to rpX-A. Only one strain was affiliated to rpX-B subgroup

Axenic culture of plant pathogenic phytoplasmas

Phytoplasmas are microorganisms associated with severe plant diseases affecting diverse agrarian activites worldwide. Since their discovery, conclusive proof of their pathogenicity is lacking due to failure to culture these organisms. This has also hindered studies on their biology, biochemistry and physiology, although significant taxonomic progress has been achieved from study of the 16S ribosomal gene and full genomic sequencing of four phytoplasma strains. The inability to fulfil Koch’s postulates severely restricts the understanding of the roles of these organisms in plant diseases and in plant/insect/phytoplasma interactions. Here we show that specific commercial media support axenic growth of phytoplasmas under defined conditions; the identity of the organisms was confirmed by PCR/RFLP analyses and sequencing of phytoplasma-specific genes. We have demonstrated for the first time that phytoplasmas, similarly to mycoplasmas, can grow independently from their host(s). This should assist reduction of the socio-economic impact of phytoplasma diseases worldwide through improved pathogen detection and consequently better management of the diseases they cause. Knowledge of mechanisms underlying the autonomous life of phytoplasmas, that are among the smallest living organisms, should also provide important information about basic mechanisms of life

Simultaneous detection of mixed ‘Candidatus Phytoplasma asteris’ and ‘Ca. Liberibacter solanacearum’ infection in carrot

Symptoms of shoot and root malformation were observed in carrot plants in fields located in the North of Gran Canaria Island (Spain), during surveys carried out in spring 2015 and 2016. Total DNA extraction from the leaves of symptomatic plants was performed and PCR assays were carried out to detect the agents possibly associated to the observed symptoms. While ‘Candidatus Liberibacter solanacearum’ was detected in the majority of tested symptomatic samples, phytoplasmas belonging to the ribosomal group 16SrI were detected only in two samples. Phylogenetic analyses and sequencing, together with virtual RFLP, confirmed that ‘Ca. L. solanacearum’ strains detected in carrot samples belong to haplotype D