Publications

4392

Citrus Huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

Huanglongbing (HLB) is a citrus infectious disease caused by ‘Candidatus Liberibacter’ spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3′-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of ‘Candidatus Liberibacter asiaticus’ (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

Huanglongbing (HLB), which is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), is a devastating disease that affects citrus trees worldwide. Because of the pervasiveness of the bacteria and psyllid vector, the disease is considered endemic in Florida. Although the effects of CLas on tree growth and physiology have been investigated for decades, most studies compared infected and noninfected trees under greenhouse conditions. This study used newly planted field-grown ‘Valencia’ sweet orange (Citrus sinensis) trees on two different rootstocks to monitor the distribution and accumulation of CLas in aboveground and belowground tissues following natural psyllid colonization and assess tree physiological responses and biomass reductions under HLB-endemic conditions. Trees were transplanted into the field with individual protective covers (IPCs), which are used to exclude psyllids and prevent infection. Openings were cut in the IPCs of half of the trees; to promote infection, these IPCs were temporarily removed during the main vegetative flushing period when psyllid populations were high. All trees that were exposed to psyllids became infected and displayed the symptoms typically associated with HLB. Throughout the study, higher levels of CLas were detected in the leaves compared with those in the fibrous roots. Trees that were not exposed to psyllids remained noninfected and healthy. After 18 months, a subset of trees was excavated to assess biomass differences between infected and noninfected trees. Infected trees had root system reductions of 37% and shoot system reductions of 20%, thereby significantly reducing the belowground-to-aboveground biomass ratio. Fibrous root loss was 49% and more severe than the loss of the rest of the root tissue. This study is the first to demonstrate the full extent of damage caused by CLas infection under natural HLB-endemic conditions. The results confirm previous observations that suggested fibrous root loss as one of the major consequences of infection and colonization with CLas. They also reinforce the benefits of using IPCs to prevent infection of young citrus trees during the first years of growth in the field.

A new disease syndrome of zucchini squash was observed in Southern Italy, in 2018 and again in 2020. Affected plants were severely stunted and leaves were bent downwards, small, stiff, thick, leathery, and had interveinal chloroses. In addition, flowers were virescent and fruits were deformed and often cracked. Disease incidence was 20 and 30% in two different zucchini cultivations in Campania region (Southern Italy). Tomato yellow leaf curl New Delhi virus (ToLCNDV) was detected in eight samples, by loop-mediated isothermal amplification–based (LAMP) kit and by PCR and Sanger sequencing of the AV1 gene. Phytoplasmas were detected in the same samples using nested PCR assays with primer pairs P1/P7 and R16F2n/R16R2. Phytoplasma associations in plant samples were confirmed using specific primers for the multilocus genes SecY, tuf and rp. Sequence comparison of multilocus genes and phylogenetic analyses of the 16S rDNA gene confirmed the association of a phytoplasma strain closely related to ‘Candidiatus Phytoplamsa asteris’. This is the first report of mixed infections of ToLCNDV and a putative ‘Ca. Phytoplamsa asteris’ strain in zucchini, associated with a new Squash-Phytoplasma-Begomovirus (SqPB) disease syndrome.

Abstract Backgroud The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated. Results The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40–80.91% and 83.98–86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus. Conclusion The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex.