Triplex real-time qPCR for the simultaneous detection of Botryosphaeriaceae species in woody crops and environmental samples

Abstract

Introduction: Species of Botryosphaeriaceae fungi are relevant pathogens of almond causing trunk cankers, extensive gumming, necrosis of internal tissues and plant dieback and dead, threatening almond productivity. A novel triplex quantitative real-time PCR (qPCR) assay was designed for the simultaneous detection and quantification of Neofusicoccum parvum, Botryosphaeria dothidea and the Botryosphaeriaceae family.

Material and methods: The method was validated in symptomatic and asymptomatic almond, avocado, blueberry and grapevine plants and in environmental samples, such as cropping soil and rainwater and in artificially inoculated trapped spores, demonstrating the same performance on several matrices.

Results and discussion: The limit of detection of the triplex qPCR was 10 fg of genomic DNA for the three fungal targets, with high correlation coefficients (R2) and amplification efficiencies between 90 and 120%. Although the triplex qPCR demonstrated to be more sensitive and accurate than the traditional plate culturing and further sequencing method, a substantial agreement (kappa index = 0.8052 ± 0.0512) was found between the two detection methods. The highly sensitive qPCR assay allows for accurate diagnosis of symptomatic plants and early detection of Botryosphaeriaceae fungi in asymptomatic plants (rootstocks and grafting scions from almond nurseries). Furthermore, the triplex qPCR successfully detected Botryosphaeriaceae fungi in environmental samples, such as cropping soils and rainwater. It was also capable of detecting as few as 10 conidia in artificially inoculated tapes. Therefore, the triplex qPCR is a valuable tool for accurate diagnosis, aiding in the implementation of suitable control measures. It enables preventive detection in asymptomatic samples, helping to avoid the introduction and spread of these pathogens in production fields. Moreover, it assists in identifying inoculum sources and quantifying inoculum levels in crop environments, contributing to a precise phytosanitary application schedule, thereby reducing production costs and preserving the environment.

Keywords: Botryosphaeria dieback; multiplex qPCR; nursery; preventive control; rainwater; soil; trapped spores; woody crops.

Figure 1

Standard curves in triplex qPCR for the quantitative and simultaneous detection of Botryosphaeria dothidea (detected with FAM fluorophore, green line), Neofusicoccum parvum (HEX fluorophore, blue line) and Botryosphaeriaceae family (Cy5 fluorophore, purple line) in different matrices: MilliQ sterile water (A), and 5 ng/ml of DNA extracted from the subcortical tissue of almond (B), avocado (C), blueberry (D) and grapevine (E) plants, respectively, as well as soil (F) and rainwater (G). Ten-fold serial dilutions of a mixture of genomic DNA from pure colonies of B. dothidea BdALM2 isolate, N. parvum NpALM2 isolate and Diplodia seriata DsALM2 isolate (1 to 10^-4 ng each) were amplified in three replicates (1, 10^-1 and 10^-2 ng standard curve points) and six replicates (10^-3 and 10^-4 ng standard curve points). Each reaction was repeated at least three times.

Figure 2

Standard curves for the quantitative detection of (A) Botryosphaeria dothidea, detected with FAM fluorophore; (B) Neofusicoccum parvum, detected with HEX fluorophore; and (C) Botryosphaeriaceae family, detected with Cy5 fluorophore under singleplex and triplex (D) qPCR assays. Serial dilutions of gDNA from pure colonies of B. dothidea, N. parvum and Diplodia seriata were amplified in three (1 ng to 10 pg) or six (1 pg to 10 fg) replicates. Efficiency (E), coefficient of determination (R²), and regression equations of standard curves are shown for each qPCR reaction. Each reaction was repeated at least three times.

Figure 3

Successful amplification of Neofusicoccum parvum present in low concentration (100 fg of gDNA) with HEX fluorophore, in the presence of high amounts (1 ng of gDNA) of Botryosphaeria dothidea (FAM fluorophore) and Diplodia seriata (Cy5 fluorophore) respectively. Replicates at each level n= 10). RFU, Relative fluorescent units.

Figure 4

Comparison of triplex qPCR and plate culturing methods for the detection of Botryosphaeriaceae fungi in 370 samples from almond, avocado, blueberry and grapevine symptomatic and asymptomatic plants.

Figure 5

Detection of serial dilutions (10 to 10⁵) of trapped spores from Neofusicoccum parvum isolate NpALM2 artificially inoculated onto glass microscope slides covered with Melinex tapes coated with silicone oil by triplex qPCR. Nine repetitions per dilution were included.