Agronomy and Crop Science

Phytoplasmas are cell-wall-less bacteria that cause diseases in approximately 1,000 plant species. ‘Candidatus Phytoplasma pyri’, the causal agent of pear decline, induces various symptoms on its hosts, leading to weakening and dieback of the plants, reduced fruit size and yield, and, consequently, considerable financial losses in all pear-growing areas. Fighting this disease requires a reliable and inexpensive method for pathogen detection in propagation material as well as plant stocks in orchards and breeding facilities. Here, we present a field-suitable detection protocol for ‘Ca. P. pyri’ based on loop-mediated isothermal amplification (LAMP) targeting the phytoplasmal 16S ribosomal DNA sequence. The combination of a simplified sample preparation method based on sodium hydroxide and colorimetric visualization of LAMP results enables a laboratory-independent pathogen detection. The detection limit is comparable with analysis by polymerase chain reaction; however, the pear decline LAMP detection method is superior in terms of ease of use, cost, and time effectiveness for obtaining results.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ‘Candidatus Liberibacter asiaticus’ and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ‘Ca. L. asiaticus’ inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ‘Ca. L. asiaticus’. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ‘Ca. L. asiaticus’ transmission by ACP. In this study, we report a new method for targeted early detection of ‘Ca. L. asiaticus’ in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. We take advantage of the fact that ‘Ca. L. asiaticus’ remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ‘Ca. L. asiaticus’ in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ‘Ca. L. asiaticus’ inoculum removal and preventing HLB from spreading.

AbstractThe relationship between Bactrocera oleae (Rossi 1790) and its endosymbiont Candidatus Erwinia dacicola is important to achieving effective control of the olive fly population in the field. This bacterium plays a crucial role in the life of B. oleae and is necessary for its fitness. Thus, in the absence of the endosymbiont, B. oleae wild populations in the field might decrease considerably. Copper is one of the most used antimicrobials for horticultural crops worldwide, and its efficacy against Ca. E. dacicola has been demonstrated in field trials. Propolis is another natural antimicrobial compound largely used for its activity in several fields. If propolis and copper prove to be efficient against wild populations of the endosymbiont B. oleae in the field, such a biological restraint might improve sustainable agriculture. We evaluated, under laboratory conditions, the effect of two different copper products (at two different concentrations, 5% and 20%) and propolis on the content of Ca. E. dacicola in the eggs and in the adult oesophageal bulbs of B. oleae. Bulbs were extracted twice, after 2 and 5 weeks of exposure. Real‐time PCR on the bulbs showed a reduction in Ca. E. dacicola content in flies treated with copper (at both 5% and 20%), and from the first to the second extraction, while flies treated with propolis showed an increment of the relative abundance of Ca. E. dacicola. Both copper products (5% and 20%) reduced the egg production after 2 and 5 weeks in comparison with the control and propolis treatments. Moreover, adult mortality was significantly higher with propolis compared with the other treatments. Thus, our results encourage further research in order to develop new tools for the control of the olive fly in the framework of an integrated pest management strategy.

‘ Candidatus Liberibacter asiaticus’ ( CL as) is associated with the most destructive disease of citrus, huanglongbing ( HLB ). The most widely used methods for detection of CL as are PCR ‐based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR ( TPE ‐ qPCR ) was compared to DNA extraction of plant tissue followed by PCR (X‐ PCR ) by testing the same tissue samples. The former estimated a higher CL as population in tissue prints than the latter ( t ‐test; P  =   0.009). All extracts prepared for TPE ‐ qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE ‐ qPCR and immunoassay. Although the detection rate by TPE ‐ qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE ‐ qPCR . Thus, a higher detection rate would be achieved by combining TPE ‐ qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CL as infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CL as preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.