Raman Spectroscopy vs Quantitative Polymerase Chain Reaction In Early Stage Huanglongbing Diagnostics

Abstract

Raman spectroscopy (RS) is an emerging analytical technique that can be used to develop and deploy precision agriculture. RS allows for confirmatory diagnostic of biotic and abiotic stresses on plants. Specifically, RS can be used for Huanglongbing (HLB) diagnostics on both orange and grapefruit trees, as well as detection and identification of various fungal and viral diseases. The questions that remain to be answered is how early can RS detect and identify the disease and whether RS is more sensitive than qPCR, the "golden standard" in pathogen diagnostics? Using RS and HLB as case study, we monitored healthy (qPCR-negative) in-field grown citrus trees and compared their spectra to the spectra collected from healthy orange and grapefruit trees grown in a greenhouse with restricted insect access and confirmed as HLB free by qPCR. Our result indicated that RS was capable of early prediction of HLB and that nearly all in-field qPCR-negative plants were infected by the disease. Using advanced multivariate statistical analysis, we also showed that qPCR-negative plants exhibited HLB-specific spectral characteristics that can be distinguished from unrelated nutrition deficit characteristics. These results demonstrate that RS is capable of much more sensitive diagnostics of HLB compared to qPCR. Plant diseases can be caused by a variety of different pathogens, such as viruses, fungi and bacteria. Huanglongbing (HLB), or citrus greening, is a devastating disease that plagues citrus trees in Asia, Africa, and more recently the Americas 1. In the United States, HLB is associated with Candidatus Liberibacter asiaticus (CLas), a gram-negative bacterium that inhabits the plant phloem in uneven and variable titers 2,3. Limited knowledge is available on CLas bacterium due to our inability to culture it in the laboratory. The bacterium is transmitted by the highly mobile Asian citrus psyllid (Diaphorina citri) enabling proliferation of HLB on large agricultural areas. The disease is also able to spread by grafting. In late stages, HLB-infected plants have asymmetric mottling on the leaves and lopsided and green fruits. HLB causes fruit drop and poor fruit quality, e.g. smaller size, less juice yield, higher acidity and lower sugar content. Early stages of the disease, however, have minimal to no symptoms and the bacteria can remain undetectable even by quantitative polymerase chain reaction (qPCR) in the trees for several years after initial infection 4. This, wherein, lies a significant challenge in regard to HLB management. Farmers are reluctant to remove trees that have normal, healthy appearances and keep producing fruits. However, if such trees have CLas bacteria, they allow for psyllids to quickly spread this devastating disease over large distances. Therefore, it becomes critical to detect HLB at the early (pre-symptomatic) stage and develop appropriate intervention strategies for HLB containment. In disease diagnostics, the most predominant methods used to confirm a pathogen are PCR and/or enzyme-linked immunosorbent as...

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