Starch Hydrolysis and Vessel Occlusion Related to Wilt Symptoms in Olive Stems of Susceptible Cultivars Infected by Verticillium dahliae

Abstract

This study investigated starch content, amount of pathogen DNA and density of occluded vessels in healthy and Verticillium dahliae infected olive shoots and stems. Starch hydrolysis is considered a mechanism to refill xylem vessels that suffered cavitation by either, drought conditions or pathogen infections. The main objective of this work was to evaluate this mechanism in olive plants subjected to V. dahliae infection or to drought conditions, in order to know the importance of cavitation in the development of wilting symptoms. In initial experiments starch content in the shoots was studied in trees of cultivars differing in the level of resistance growing in fields naturally infested with V. dahliae. The starch content, esteemed by microscopic observation of stem transversal sections stained with lugol, decreased with the level of symptom severity. Results were confirmed in a new experiment developed with young plants of cultivars 'Picual' (highly susceptible), 'Arbequina' (moderately susceptible) and 'Frantoio' (resistant), growing in pots under greenhouse conditions, either inoculated or not with V. dahliae. In this experiment, the pathogen DNA content, quantified by real-time PCR, and the density of occluded vessels, recorded by microscopic observations of transversal sections stained with toluidine blue, were related to the symptoms severity caused by the pathogen. Finally, a drought experiment was established with young plants of the cultivar 'Picual' grown in pots under greenhouse conditions in order to compare the effects caused by water deficit with those caused by the pathogen infection. In both cases, results show that starch hydrolysis occurred, what indirectly evidence the importance of xylem cavitation in the development of the symptoms caused by V. dahliae but in the water stressed plants no vessel occlusion was detected.

Keywords: Olea europaea; Verticillium wilt; defoliating pathotype; drought stress; xylem cavitation.

FIGURE 1

(A) Transverse section of an olive stem stained with Lugol showing starch accumulation. Cells with starch accumulation, stained black inside, are abundant in the pith, axial, and radial systems of both the xylem and phloem, and in the cortex, but they are absent in the suber. (B) Xylem in the transverse section of an olive stem stained with toluidine blue showing occlusions in the vessels. Non-occluded vessels, such as 1, can be distinguished from occluded vessels, such as 2.

FIGURE 2

Starch content in different stem tissues of olive shoots (2012 experiment on naturally infested fields). The shoots were collected from trees without symptoms and from asymptomatic or dead zones of trees affected by Verticillium dahliae during field surveys in 2012, and starch content was assessed according to a 0-to-3 scale using a microscope. The values represent the mean of 10 shoots and columns with different letters in each tissue are significantly different according to Fisher’s protected LSD test at P = 0.05. Bars represent the mean (±) standard errors.

FIGURE 3

Starch content in different stem tissues and the density of occluded vessels (inside figure) of olive shoots (2013 experiment). Asymptomatic (n = 7), affected (n = 21), and dead (n = 4) shoots were collected in an olive orchard of the ‘Picual’ cultivar affected by V. dahliae during field surveys conducted in 2013. Starch content was assessed according to a 0-to-3 scale using a microscope. Density of occluded vessels was quantified using a microscope. Columns with different letters in each tissue were significantly different according to Fisher’s protected LSD test at P = 0.05. Bars represent the mean (±) standard errors.

FIGURE 4

Progress of disease severity and amount of V. dahliae DNA in plants of ‘Picual,’ ‘Arbequina,’ and ‘Frantoio’ cultivars inoculated with V. dahliae (experiment of artificial inoculation). The disease severity values are the means of 14 replicates in each week according to a 0-to-4 scale. Columns represent the average amount of V. dahliae DNA in three sampled plants (hatched columns for “Arbequina’ and pointed columns for ‘Picual’). Different letters indicate significant differences in disease severity according to a non-parametric Kruskal–Wallis test at P = 0.05. An asterisk indicates significant difference in the amount of DNA between ‘Arbequina’ and ‘Picual’ according to according to Fisher’s protected LSD test at P = 0.05. Bars represent the mean (±) standard errors.

FIGURE 5

Starch content in different stem tissues of ‘Picual’ (A), ‘Arbequina’ (B), and ‘Frantoio’ (C) cultivars not inoculated or inoculated with V. dahliae (experiment of artificial inoculation). Three groups of plants were compared: non-inoculated (n = 15), inoculated with Disease Severity (DS) < 1 (n = 4 for ‘Picual,’ n = 5 for ‘Arbequina,’ and n = 15 for ‘Frantoio’) and inoculated with Disease Severity (DS) ≥ 1 (n = 11 for ‘Picual,’ n = 10 for ‘Arbequina,’ and n = 0 for ‘Frantoio’). Starch content was assessed according to a 0-to-3 scale using a microscope. Columns with different letters in each tissue are significantly different according to Fisher’s protected LSD test at P = 0.05. Bars represent the mean (±) standard errors.

FIGURE 6

Density of occluded vessels in plants of ‘Picual,’ ‘Arbequina,’ and ‘Frantoio’ cultivars non-inoculated or inoculated with V. dahliae (experiment of artificial inoculation). Plants are grouped according to the level of disease severity shown: non-inoculated, inoculated with Disease Severity (DS) < 1 and inoculated with Disease Severity (DS) ≥ 1. Density of occluded vessels was quantified using a microscope. Columns with different letters for each cultivar are significantly different according to Fisher’s protected LSD test at P = 0.05. Bars represent the mean (±) standard errors.

FIGURE 7

Starch content in the different stem tissues of plants of ‘Picual’ cultivar subjected to three different irrigation treatments (drought stress experiment). Values are the mean of 5, 6, and 4 plants from the irrigation treatments: non-irrigation and irrigation after drought, respectively. Columns with different letters in each tissue are significantly different according to the Fisher’s protected LSD test at P = 0.05. Xylem vessel occlusions were not detected in any of the groups.