Plant Science

Abstract In the spring of 2012, sophora (Sophora alopecuroides L.) plants showing symptoms of leaf yellowing, little leaves and stunting were observed in Firooz-kuh (Tehran province), Sari (Mazandaran province) and Urmia (West Azerbaijan province) in Iran. Symptomatic plants from the three locations were subjected to nested polymerase chain reaction (PCR) to amplify 16SrRNA using primer pair P1/P7 followed by primer pair R16F2n/R16R2. The amplicons were purified, sequenced and the nucleotide sequences were analyzed by virtual restriction fragment length polymorphism (RFLP). The phytoplasmas associated with the yellows disease were identified as members of the 16SrIX group (Candidatus Phytoplasma phoenicium) and the 16SrXII group (Candidatus Phytoplasma solani). The two phytoplasmas were placed in 16SrIX-C and 16SrXII-A subgroups, respectively, in constructed phylogenetic trees. This is the first report on sophora yellows associated with Candidatus Phytoplasma phoenicium.

‘Candidatus Liberibacter asiaticus’ (CLas), an α-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, two cases of CLas have been detected in Los Angeles County, one in Hacienda Heights in 2012 and the other in San Gabriel in 2015. Although all infected trees were destroyed in compliance with a state mandate, citrus industry stakeholder concerns about HLB in California are high. Little is known about the biology of CLas, particularly the California strains, hindering effective HLB management efforts. In this study, next-generation sequencing technology (Illumina MiSeq) was employed to characterize the California CLas strains. Data sets containing >4 billion (Giga) bp of sequence were generated from each CLas sample. Two prophages (P-HHCA1-2 and P-SGCA5-1) were identified by the MiSeq read mapping technique referenced to two known Florida CLas prophage sequences, SC1 and SC2. P-HHCA1-2 was an SC2-like or Type 2 prophage of 38,989 bp in size. P-SGCA5-1 was an SC1-like or Type 1 prophage of 37,487 bp in size. Phylogenetic analysis revealed that P-HHCA1-2 was part of an Asiatic lineage within the Type 2 prophage group. Similarly, P-SGCA5-1 was part of an Asiatic lineage within Type 1 prophage group. The Asiatic relatedness of both P-HHCA1-2 and P-SGCA5-1 was further presented by single nucleotide polymorphism analysis at terL (encoding prophage terminase) that has been established for CLas strain differentiation. The presence of different prophages suggests that the two California CLas strains could have been introduced from different sources. An alternative explanation is that there was a mixed CLas population containing the two types of prophages, and limited sampling in a geographic region may not accurately depict the true CLas diversity. More accurate pathway analysis may be achieved by including more strains collected from the regions.

Zebra chip (ZC) disease, a serious threat to the potato industry, is caused by the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso). Five haplotypes (hapA to hapE) of this pathogen have been described so far in different crops, with only hapA and hapB being associated with ZC in potato. Both haplotypes are vectored and transmitted to a variety of solanaceaeous plants by the tomato/potato psyllid, Bactericera cockerelli (Šulc). Psyllids are native to North America, and four haplotypes have been identified and named based on their predominant geographic association: Northwestern, Central, Western, and Southwestern. Although all psyllid haplotypes have been found in southern Idaho potato fields, data on relative haplotype abundances and dynamic changes in the fields over time have not previously been reported. Here, psyllid samples collected in Idaho potato fields from 2012 to 2015 were used to clarify spatial and temporal patterns in distribution and abundance of psyllid and Lso haplotypes. A shift from hapA toward hapB population of Lso was revealed during these four seasons, indicating possible evolution of Lso in Idaho fields. Although we confirmed that Western psyllids were the most abundant by far during the four seasons of observation, we also observed changes in abundance of other haplotypes, including increased diversity of psyllid haplotypes during 2015. Seasonal changes observed for the Northwestern and Central haplotypes could potentially be linked to psyllid migration and/or habitat changes. South-central Idaho exhibited more diversity in psyllid haplotypes than southwestern Idaho.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely ‘Candidatus Liberibacter asiaticus’ (Las), ‘Candidatus Liberibacter africanus’, and ‘Candidatus Liberibacter americanus’. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

A recently described symbiosis between the metabolically streamlined nitrogen‐fixing cyanobacterium UCYN‐A and a single‐celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN‐A sublineages have been defined based on UCYN‐A nitrogenase (nifH) sequences. Due to the low abundances of UCYN‐A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN‐A sublineages based on high throughput sequencing of UCYN‐A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN‐A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN‐A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN‐A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN‐A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN‐A distributions and the ecological importance of the different sublineages.

The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) region of Minas Gerais State and the south of São Paulo State (SPS). Significant climatic variation occurs between regions which could affect huanglongbing (HLB) progress, which is lower in TM. To investigate this possibility, young sweet orange shoots were sampled periodically over 2 years to determine ‘Candidatus Liberibacter asiaticus’ titers in naturally infected trees in orchards in Analândia, central SPS, and Frutal and Comendador Gomes, within TM. Data-loggers recorded local temperature and relative humidity hourly. In the laboratory, five ‘Ca. L. asiaticus’-free Diaphorina citri adults were placed on each sampled shoot for 48 h to feed and acquire the pathogen. Shoots and insects were individually analyzed by quantitative polymerase chain reaction to determine ‘Ca. L. asiaticus’ titers. The incidence of ‘Ca. L. asiaticus’-positive shoots, ‘Ca. L. asiaticus’ titers, and acquisition rates were lower for shoots from Comendador Gomes than those from Frutal or Analândia. Stronger association was observed between ‘Ca. L. asiaticus’ titers and the number of hours below 15°C (h < 15°C) or above 30°C (h > 30°C), and cumulative rainfall registered during the 15 days prior to sampling of shoots on each occasion. ‘Ca. L. asiaticus’ titers associated positively with h < 15°C and rainfall and negatively with h > 30°C. The slower spread and lower incidence of HLB in TM may be related to lower incidences of ‘Ca. L. asiaticus’-positive young shoots and lower titers of ‘Ca. L. asiaticus’ in the same shoots as a consequence of the warmer and drier conditions.

Although ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen associated with citrus huanglongbing (HLB), some characteristics of transmission by the psyllid vector Diaphorina citri are not fully understood. We examined the latent period and persistence of transmission of Las by D. citri in a series of experiments at 25°C, in which third-instar psyllid nymphs and 1-week-old adults were confined on infected citrus for an acquisition access period (AAP), and submitted to sequential inoculation access periods (IAPs) on healthy citrus seedlings. The median latent period (LP50, i.e., acquisition time after which 50% of the individuals can inoculate) of 16.8 and 17.8 days for psyllids that acquired Las as nymphs and adults, respectively, was determined by transferring single individuals in 48-h IAPs. Inoculation events were intermittent and randomly distributed over the IAPs, but were more frequent after acquisition by nymphs. A minimum latent period of 7 to 10 days was observed by transferring groups of 10 psyllids in 48-h IAPs, after a 96-h AAP by nymphs. Psyllids transmitted for up to 5 weeks, when submitted to sequential 1-week IAPs after a 14-day AAP as nymphs. The long latent period and persistence of transmission are indirect evidences of circulative propagation of Las in D. citri.

Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Brazil, is associated mainly with the bacterium ‘Candidatus Liberibacter asiaticus’ transmitted by the psyllid Diaphorina citri. The aim of this study was to improve our understanding of the ‘Ca. L. asiaticus’ infection process by exposing excised, fully expanded, immature citrus leaves in 50-ml Falcon tubes to one, four, or eight adults from a ‘Ca. L. asiaticus’-exposed colony for 1-, 3-, 7-, or 15-day periods for access to inoculation (IAP). The leaves were incubated at 26°C for 1, 3, 7, 15, and 21 days (incubation period [IP]). Infection frequencies and ‘Ca. L. asiaticus’ titers were assessed by quantitative polymerase chain reaction (qPCR). ‘Ca. L. asiaticus’ infection was a function of leaf age, number of insects, IAP, and IP. In general, higher infection rates were observed on younger leaves inoculated with higher numbers of insects and after longer IAP and IP. The immature excised leaf method allowed determination of 3 to 7 days as the range of time required by ‘Ca. L. asiaticus’ to reach qPCR detectable levels. Even though leaf survival could be prolonged by the maintenance of a branch segment at the base of the leaf petiole, leaf degradation, visible after about 15 days IP, did not allow observation of the entire infection process which, in the intact plant, culminates with the appearance of the blotch mottling symptom on leaf blades.