Horticulture

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.