Understanding the Process of Latent Infection of Canker-Causing Pathogens in Stone Fruit and Nut Crops in California

Abstract

The Botryosphaeriaceae family is considered a fungal family that includes pathogens causing latent infection of woody plants, and a number of species were identified as causal pathogens of canker and shoot blight diseases. To better understand the process of latent infection of major canker-causing pathogens in woody tissues in different tree crops important in California, shoot and bud samples were randomly collected from four tree crops: almond, dried plum, pistachio, and walnut. The previously developed DNA primers and quantitative real-time PCR (qPCR) assay systems were applied to detect six canker-causing pathogen groups, including Botryosphaeria dothidea, and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis. The concepts of molecular severity (MS) and latent infection index (LII) were introduced and applied to quantify the latent infection levels for these samples. Variation in incidence of latent infection among pathogen groups was observed, whereas the incidences were relatively low among species of Phomopsis and Diplodia. High incidences of Cytospora spp. were observed in two dried plum (prune) orchards. Most orchards showed high incidences of B. dothidea and Lasiodiplodia spp. and moderate incidences of Neofusicoccum spp. Variations in MS were observed among samples of the studied orchards, ranging from 4 to 8. The overall results of LII demonstrated that species of Diplodia and Phomopsis were less important in population development of canker-causing pathogens at the latent phase. Lasiodiplodia spp. were the most aggressive and had been well developed in populations among the studied tree crops. Cytospora spp. became predominant in two of the three dried plum orchards, whereas B. dothidea and Neofusicoccum spp. showed trends of increase in incidence across various tree crops. This study also demonstrated the usefulness of this sensitive qPCR approach in providing evidence of the latent phase of major canker-causing pathogens of stone fruit and nut crops at an early stage of latent infection in woody plant tissues.

Inoculum quantification of canker‐causing pathogens in prune and walnut orchards using real‐time PCR

“…Unlike the regular standard curves describing a relationship between DNA amount and Ct value (DNA-Ct) as most real-time PCR assay used (Bustin et al 2009), to reasonably consider the unavoidable spore losses during sample extraction and to properly quantify the spore concentration in real samples of air or rainwater, we used the standard curves directly describing the relationship between spore concentration and Ct value, for which the serial dilutions of spore samples were generated before DNA extraction. Although the results showed reduced R 2 value compared with that of DNA-Ct curve, these standard curves are more useful and practical in application of processing real samples, and results were more accurate to represent real situations in orchards as shown in our previous studies (Luo et al 2017(Luo et al , 2019.…”

“…and Diplodia spp (Table 1). These primers were once successfully used in a real-time quantitative PCR (qPCR) system to quantify the latent infection levels from shoot tissues of different crops (Luo et al 2019).…”

“…The protocol for DNA extraction from spore suspension described above was applied to obtain the template DNA Table 1 The equations of standard curves obtained in this study to quantify the spore concentrations for the samples from rainwater and air in prune and walnut orchards. The sequences of primers were from a previous study (Luo et al 2017(Luo et al , 2019 Pathogen group The approximate minimal number of spores that the assay can detect in each PCR reaction in the corresponding 2 ll of template DNA. ‡…”

“…Lasiodiplodia, Neofusicoccum and Phomopsis (Luo et al 2017). We also developed a qPCR assay to quantify the levels of latent infection by these pathogen groups in plant tissues of stone fruit and nut crops (Luo et al 2017(Luo et al , 2019. This assay was successfully used to conduct a survey on incidence of latent infection by these pathogen groups in almond, prune, pistachio and walnut orchards in California (Luo et al 2019).…”

“…We also developed a qPCR assay to quantify the levels of latent infection by these pathogen groups in plant tissues of stone fruit and nut crops (Luo et al 2017(Luo et al , 2019. This assay was successfully used to conduct a survey on incidence of latent infection by these pathogen groups in almond, prune, pistachio and walnut orchards in California (Luo et al 2019). Finding that different pathogen groups were predominant on different tree crops was very useful information to understand each specific situation of latent infection and the potential risk of canker development and pathogen population dynamics in the future.…”