Plant Pathology

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘ Candidatus Liberibacter asiaticus’ and ‘ Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘ Ca.  L. asiaticus’, which has taken over from ‘ Ca.  L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘ Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘ Ca.  L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘ Ca.  L. asiaticus’ was distributed throughout the inoculated plants, with mean C t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘ Ca.  L. asiaticus’ reached the highest titres in mature leaves (mean C t value = 26·7) of citrus plants maintained at 22/27°C. ‘ Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C t value in mature leaves = 33·6).

The presence of phytoplasmas in seven coniferous plant species ( Abies procera , Pinus banksiana , P. mugo , P. nigra , P. sylvestris , P. tabuliformis and Tsuga canadensis ) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with Alu I, Mse I and Rsa I endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘ Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘ Candidatus Phytoplasma pini’, group 16SrXXI.

The aim of this work was to assess the effects of a combined inoculum of a rhizobacterium and an arbuscular mycorrhizal (AM) fungus on plant responses to phytoplasma infection, and on phytoplasma multiplication and viability in Chrysanthemum carinatum plants infected by chrysanthemum yellows phytoplasma (CY). Combined inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif resulted in some resistance to phytoplasma infection (about 30%), delayed symptom expression in nonresistant plants, improved growth of the aerial part of the infected plants (+68·1%), and altered root morphology (root tip number: +49·9%; branching degree: +82·8%). Combined inoculation with the two beneficial microorganisms did not alter CY multiplication and viability. In inoculated and infected plants, phytoplasma morphology was typical of senescent cells. A more active and efficient root system in double‐inoculated plants probably mediated the effects of the two rhizospheric microorganisms in the infected plants. The practical application of rhizospheric microorganisms for mitigating phytoplasma damage, following evaluation under field conditions, represents an additional tool for the integrated management of phytoplasmosis.

Elm yellows phytoplasmas (EY) belonging to the 16SrV‐A subgroup were recently proposed as a new candidate species ‘ Candidatus Phytoplasma ulmi’. These pathogens infect elm trees, causing leaf yellowing and premature drying. In this study, 25 isolates originating from localities in northeast, east and southwest Serbia were characterized by means of RFLP analysis and DNA sequencing of four genomic loci: 16S rRNA, ribosomal protein rpl22 ‐ rps3 , secY and map . In total, five different genotypes were identified based on collective sequencing of all four genes. Four of these genotypes showed significant nucleotide changes compared with the EY1 T reference strain. Phylogeny based on parsimony analyses of ribosomal protein, secY and map genetic loci indicated a single monophyletic origin of EY1 T and the new ‘ Ca . Phytoplasma ulmi’ strains. Unlike phylogenetic clustering, DNA sequence comparison of EY1 T and the novel strains revealed mutations in oligonucleotide signature sequences for all three genes (16S, rpl22 ‐ rps3 and secY ) used for the characterization and assignment of 16SrV‐A phytoplasmas to the ‘ Ca.  Phytoplasma ulmi’ species in the original description. Based on their high degree of genetic variability, the Serbian strains were assigned to four different subtypes of ‘ Ca.  Phytoplasma ulmi’ (EY‐S1, EY‐S2, EY‐S3 and EY‐S4). New diagnostic enzymes for practical use in ‘ Ca.  Phytoplasma ulmi’ identification are proposed for the 16S rRNA, ribosomal protein and secY genes. The implications of genetic variability within signature sequences for taxonomy and identification of ‘ Ca . Phytoplasma’ species, as well as the importance of geographic variability and number of strains characterized for species description, are discussed.

Changes in carbohydrate metabolism in sweet orange ( Citrus sinensis ) infected with ‘ Candidatus Liberibacter asiaticus’, a purported cause of citrus Huanglongbing (HLB), were investigated. Starch levels in HLB‐infected leaves with and without symptoms increased 3·1‐ and 7·9‐fold, respectively, compared to healthy controls. In symptomless leaves, sucrose and fructose accumulated significantly ( P  <   0·05) in both midribs and lobes, and glucose only in the midribs (greater than fivefold); whereas maltose levels were reduced to 64·6% and 86·8% in the midribs and foliar lobes, respectively, of the values in healthy leaves. In leaves with symptoms, sucrose and glucose remained at high levels compared to healthy leaves, whilst no accumulation of fructose was observed; by contrast, the maltose content decreased to as low as 49·6% of that in healthy leaves. Fourfold induction of cell‐wall‐bound invertase activity was detected in both types of leaves on diseased plants. Additionally, the expression profiles of starch breakdown genes suggested that the transcription of DPE2 and MEX1 was down‐regulated. Together with the reduction of maltose, it is suggested that the impairment of starch breakdown may contribute to the starch accumulation in infected leaves. The imbalance of carbohydrate partitioning and its relation to disease physiology are discussed.