Publications

3884

The knowledge of phytoplasma genetic variability is a tool to study their epidemiology and to implement an effective monitoring and management of their associated diseases. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” disease in grapevines, and yellowing and decline symptoms in many plant species, causing serious damages during the epidemic outbreaks. The epidemiology of the diseases associated with this phytoplasma is complex and related to numerous factors, such as interactions of the host plant and insect vectors and spreading through infected plant propagation material. The genetic variability of ‘Ca. P. solani’ strains in different host species and in different geographic areas during the last two decades was studied by RFLP analyses coupled with sequencing on vmp1, stamp, and tuf genes. A total of 119 strains were examined, 25 molecular variants were identified, and the variability of the studied genes was linked to both geographic distribution and year of infection. The crucial question in ‘Ca. P. solani’ epidemiology is to trace back the epidemic cycle of the infections. This study presents some relevant features about differential strain distribution useful for disease monitoring and forecasting, illustrating and comparing the phytoplasma molecular variants identified in various regions, host species, and time periods.

Florida citrus production has declined 75% due to Huanglongbing (HLB), a disease caused by the pathogenic bacterium Candidatus Liberibacter asiaticus (CLas). Methods to combat CLas are costly and only partially effective. The cross-compatible species Poncirus trifoliata and some of its hybrids are known to be highly tolerant to CLas, and thus can potentially serve as an alternative feedstock for many citrus products. To further investigate the commercial potential of citrus hybrids, three citrus hybrids, US-802, US-897, and US-942, were studied for their potential as feedstocks for citrus co-products using steam explosion (STEX) followed by water extraction. Up to 93% of sugars were recovered. US-897 and US-942 have similar volatile profiles to that of the commercial citrus fruit types and as much as 85% of these volatiles could be recovered. Approximately 80% of the pectic hydrocolloids present in all three hybrids could be obtained in water washes of STEX material. Of the phenolics identified, the flavanone glycosides, i.e., naringin, neohesperidin, and poncirin were the most abundant quantitatively in these hybrids. The ability to extract a large percentage of these compounds, along with their inherent values, make US-802, US-897, and US-942 potentially viable feedstock sources for citrus co-products in the current HLB-blighted environment.

A novel actinobacterium, strain YIM 96748T, was isolated from a saline soil sample collected from the south bank of Aiding Lake in Xinjiang Province, Northwest China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM 96748T is closely related to Amycolatopsis cihanbeyliensis BNT52T (98.9%) and Amycolatopsis jiangsuensis KLBMP 1262T (97.2%). The DNA–DNA relatedness between strain YIM 96748T and its closest type strain A. cihanbeyliensis BNT52T was 59.6%. The average nucleotide identity between strain YIM 96748T and its neighbor strain was 88.97%. Based on the genotypic and phenotypic characteristics, it is concluded that strain YIM 96748T represents a novel species of the genus Amycolatopsis, whose name was proposed as Amycolatopsis aidingensis sp. nov. The type strain is YIM 96748T. To investigate the biosynthetic potential of producing secondary metabolites, the complete genome of YIM 96748T was sequenced and analyzed. The complete genome sequence of YIM 96748T consists of a 7,657,695-bp circular chromosome, comprising 7,162 predicted genes with a DNA G + C content of 70.21 mol%. Fifty-one putative biosynthetic gene clusters of secondary metabolites were found, including the antibacterial/antitumor agent TLN-05220, the antibacterial agent nocardicin A, the antifungal agent nystatin A1, and the osmolyte ectoine. The investigation of the secondary metabolites of A. aidingensis YIM96748T led to the discovery of two new phenylpropyl acetate enantiomers, amycoletates A (1) and B (2), and five known compounds: 4-hydroxy phenethyl acetate (3), 2-p-acetoxyphenylethanol (4), (S)-ethyl indole-3-lactate (5), (R)-ethyl indole-3-lactate (6), and p-hydroxybenzoic acid (7). One of the gene clusters 14, 36, and 43, which contain a single module of polyketide synthase, might be responsible for the biosynthesis of compounds 1 and 2 from compound 7 as a precursor. Further studies, including the one strain many compounds approach (OSMAC) and genetic modification, are needed to explore novel compounds from this talented halophilic Amycolatopsis strain.

In sweet cherry (Prunus avium), infection by ‘Candidatus Phytoplasma pruni’ results in small fruit with poor color and taste, rendering the fruit unmarketable. Yet the disease pathology is poorly understood, particularly at the cultivar level. Therefore, in this study we examined the physiological effects of Ca. P. pruni infection across a range of cultivars and locations in eastern Washington. We found that infection could be separated into early and established stages based on pathogen titer, which correlated with disease severity, including fruit size, color, and sugar and metabolite content. Furthermore, we observed that the effects of early-stage infections were largely indistinguishable from healthy, uninfected plants. Cultivar- and location-specific disease outcomes were observed with regard to size, color, sugar content, and citric acid content. This study presents the first in-depth assessment of X-disease symptoms and biochemical content of fruit from commercially grown sweet cherry cultivars known to be infected with Ca. P. pruni.

Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, ‘Candidatus Liberibacter asiaticus’ (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid–based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.

A protocol to successfully transmit the huanglongbing (HLB) pathogen, ‘Candidatus Liberibacter asiaticus’, between citrus plants by using the Asian citrus psyllid (ACP) and an alternative way to help growers control ACP are proposed. Best results were obtained when pathogen acquisition by adults reared on fully symptomatic ‘Ca. Liberibacter asiaticus’-positive plants, latency, and inoculation occurred at ambient air temperatures ranging from 24 to 28°C and when a single infective adult ACP was confined for 7 days on soft, newly developing vegetative shoots (stages v2 to v4). No infection resulted from confinement of infective ACP adults on mature leaves (stage v6). Under the described conditions, single ACP adults could successfully transmit ‘Ca. Liberibacter asiaticus’ to an average of 56.5% (35 to 83%) of plantlets with v2 to v4 shoots growing in 0.3-liter tubes and to 80.5% (76 to 86%) of plants with v2 to v4 shoots growing in 4.7-liter pots. The use of single insects and plantlets reduces labor, space, and other resources necessary to undertake transmission tests. It also reduces time needed for transmission studies and should help accelerate research on HLB. The results were used to develop an index for favorability to infection (IFI) to determine orchard vulnerabilities to ‘Ca. Liberibacter asiaticus’. The IFI is based on the heterogeneous population of new shoots that occurs on tree canopies and may offer alternative or complementary alternatives to the laborious and costly insect surveys currently used in most instances to determine threshold levels for insecticide applications.