Plant Science

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘ Candidatus Phytoplasma pyri’, but none were infected by the ‘ Ca . Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘ Ca . Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘ Ca . Phytoplasma pyri’ phytoplasma was Cacopsylla pyri . Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘ Ca . Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘ Ca . Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.

Huanglongbing (HLB; “citrus greening” disease) has caused significant damages to the global citrus industry as it has become well established in leading citrus-producing regions and continues to spread worldwide. Insecticidal control has been a critical component of HLB disease management, as there is a direct relationship between vector control and Candidatus Liberibacter spp. (i.e., the HLB pathogen) titer in HLB-infected citrus trees. In recent years, there have been substantial efforts to develop practical strategies for specifically managing Ca. Liberibacter spp.; however, a literature review on the outcomes of such attempts is still lacking. This work summarizes the greenhouse and field studies that have documented the effects and implications of chemical-based treatments (i.e., applications of broad-spectrum antibiotics, small molecule compounds) and nonchemical measures (i.e., applications of plant-beneficial compounds, applications of inorganic fertilizers, biological control, thermotherapy) for phytopathogen control. The ongoing challenges associated with mitigating Ca. Liberibacter spp. populations at the field-scale, such as the seasonality of the phytopathogen and associated HLB disease symptoms, limitations for therapeutics to contact the phytopathogen in planta, adverse impacts of broad-spectrum treatments on plant-beneficial microbiota, and potential implications on public and ecosystem health, are also discussed.

Prophages, the lysogenic form of bacterial phages, are important genetic entities of ‘Candidatus Liberibacter asiaticus’ (CLas), a nonculturable α-proteobacterium associated with citrus Huanglongbing. Two CLas prophages have been described, SC1 (NC_019549.1, Type 1) and SC2 (NC_019550.1, Type 2), which involve the lytic cycle and the lysogenic cycle, respectively. To explore the prophage repertoire, 523 CLas DNA samples extracted from leaf petioles of CLas-infected citrus were collected from southern China and surveyed for Type 1 and Type 2 prophages by specific PCR. Eighteen samples were found lacking both prophages. One sample, JXGC, sequenced using Illumina HiSeq, generated >100 million short sequence reads (150 bp per read). Read mapping to known prophage sequences showed a sequence coverage of 46% to SC1 and 50% to SC2. BLAST search using SC1 and SC2 as queries identified three contigs from the JXGC de novo assembly that form a circular P-JXGC-3 (31,449 bp), designated as a new Type 3 prophage. Chromosomal integration of P-JXGC-3 was detected to occur within a helicase gene, resulting in a duplication of this gene. P-JXGC-3 had 36 open reading frames (ORFs), 10 of which were not found in Type 1 or Type 2 prophages, including four genes that encoded a restriction-modification (R-M) system (hsdR, hsdS, hsdM1, and hsdM2). Typed by prophage-specific PCR, the CLas strains in southern China contained all combinations of the three prophage types with the exception of a Type 2−Type 3 combination, suggesting active ongoing prophage−phage interactions. Based on gene annotation, P-JXGC-3 is not capable of reproduction via the lytic cycle. The R-M system was speculated to play a role against Type 1 prophage−phage invasion.

The nonculturable bacterium ‘Candidatus Liberibacter solanacearum’ is the causative agent of zebra chip disease in potato. Computational analysis of the ‘Ca. L. solanacearum’ genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to serralysin. Serralysin and other serralysin family metalloprotease are typically characterized as virulence factors and are secreted by the type I secretion system (T1SS). The ‘Ca. L. solanacearum’ serralysin-like gene is located next to and divergently transcribed from genes encoding a T1SS. Based on its relationship to the T1SS and the role of other serralysin family proteases in circumventing host antimicrobial defenses, it was speculated that a functional ‘Ca. L. solanacearum’ serralysin-like protease could be a potent virulence factor. Gene expression analysis showed that, from weeks 2 to 6, the expression of the ‘Ca. L. solanacearum’ serralysin-like gene was at least twofold higher than week 1, indicating that gene expression stays high as the disease progresses. A previously constructed serralysin-deficient mutant of Serratia liquefaciens FK01, an endophyte associated with insects, as well as an Escherichia coli lacking serralysin production were used as surrogates for expression analysis of the ‘Ca. L. solanacearum’ serralysin-like gene. The LsoA and LsoB proteins were expressed as both intact proteins and chimeric S. liquefaciens-‘Ca. L. solanacearum’ serralysin-like proteins to facilitate secretion in the S. liquefaciens surrogate and as intact proteins or as a truncated LsoB protein containing just the putative catalytic domains in the E. coli surrogate. None of the ‘Ca. L. solanacearum’ protein constructs expressed in either surrogate demonstrated proteolytic activity in skim milk or zymogram assays, or in colorimetric assays using purified protein, suggesting that the ‘Ca. L. solanacearum’ serralysin-like gene does not encode a functional protease, or at least not in our surrogate systems.

Zebra chip (ZC) disease of potato is associated with the putative pathogen ‘Candidatus Liberibacter solanacearum’, which is transmitted by the potato psyllid Bactericera cockerelli (Hem., Triozidae). The present study was initiated to investigate ‘Ca. L. solanacearum’ development during and following typical commercial storage practices. Using bacteriliferous psyllids, Russet Norkotah potato tubers were infested in field cages 14, 10, and 4 days before harvest. Changes in ‘Ca. L. solanacearum’ detection rate, ‘Ca. L. solanacearum’ titer, and concentrations of phenolic compounds were documented throughout storage. ‘Ca. L. solanacearum’ titer continued to increase during storage. Although significant increases in the frequency of ‘Ca. L. solanacearum’ detection were observed in all infestation treatments, the impact of ‘Ca. L. solanacearum’ infection on tuber quality remained comparatively low in plants infected 4 days before harvest, because the majority of the tubers remained asymptomatic. Minimizing storage and retail chain movement durations would help to limit ‘Ca. L. solanacearum’ impact on tuber quality in tubers infected 14 and 10 days before harvest. This study also demonstrated that ‘Ca. L. solanacearum’ can relocate from a newly infected leaf to a tuber in as little as 4 days. Psyllid management is recommended until at least 4 days before green harvest, when psyllid pressure is high in fields in which tubers are destined for commercial storage.