Bulletin OEPP/EPPO Bulletin 23, 537-541 (1993)

Evaluation of the susceptibility of olive cultivars to verticillium wilt'

by A. M. PENNISI, S. 0. CACCIOLA*, G. MAGNANO D I SAN LIO* and G. PERROTTA*

Istituto di Difesa delle Piante, Universita di Reggio Calabria, Piazza S. Francesco di Sales 2, 89061 Reggio Calabria (Italy)

* Istituto di Patologia vegetale, Universiti di Catania, via Valdisavoia 5, 95123 Catania (Italy)

In the present study, we evaluated the susceptibility of different commercial olive cultivars to verticillium wilt. Two Verticillium duhliue isolates, obtained from olive and artichoke, were used in pathogenicity tests. Two-year-old rooted cuttings were inoculated using either the root-dip or the stem-wounding method. The results were similar with both inoculation methods. Cvs Carolea and Cipressino proved to be moderately susceptible whereas Cassanese, Nocellara del Belice, Nocellara Etnea, Tonda Iblea and Uovo di Piccione were very susceptible. The response of cv. Coratina varied from susceptibility to moderate susceptibility.

Introduction

Verticillium wilt of olive, caused by Verticillium duhliue, was first reported in 1946 (Ruggieri, 1946). Although epidemic outbreaks of the disease have been recorded in Central and Southern Italy (Bonifacio, 1974; Cirulli & Montemurro, 1976; Bonifacio & Parrini, 1978), it has never reached epidemic proportions in olive orchards in Sicily.

In 1990-10, V. duhliue infections were observed on container-grown 2-year-old rooted cuttings of olive cv. Carolea in a nursery near Milazzo (Sicily). About 10% of 2000 cuttings were affected. Carolea is a cultivar originating from Calabria which is being widely used in new plantings in Southern Italy and Sicily. The degree of susceptibility of this olive cultivar to verticillium wilt is not, however, known. The present study aimed at evaluating the susceptibility to V . duhliae infections of Carolea in comparison with other commercial olive cultivars.

Materials and methods

Two isolates of V. duhliue obtained from olive and artichoke, both producing microsclerotia in culture, were used in this study. They showed variable colony morphology due to their tendency to form sectors; the identification, however, was confirmed by comparing their total mycelial protein electrophoretic profiles with those of V . duhliue isolates identified previously. The electrophoretic phenotypes of subcultures obtained from variant sectors of both isolates were very similar or identical to those of isolates from which they originated (Fig. 1). Protein extractions and electrophoretic analysis were performed according to procedures described elsewhere (Cacciola & Magnano di San Lio, 1988; Granata et ul., 1992).

Inoculations were made with a conidial suspension prepared from 7-day-old cultures grown on potato-dextrose-agar (PDA) slants in Petri dishes a t 24°C in the dark. The cultures were flooded with sterile distilled water and their surface was gently scraped with a sterile scalpel. The

' Paper presented at the Joint MPU/EPPO Conference on Olive Diseases, Sounion (GR), 1992-05-05/08.

537

538 A . M . Pennisi et al.

Fig. 1. Electrophoretic profiles of total mycelial proteins of Vwticilliurn duhliae isolates on SDS polyacrylamide gradient gel (10-20%). Gel stained with Coomassie blue. The samples on the left of the gel contained 35 pg of proteins each whereas the wells on the right were loaded with 50 p g of proteins. Profiles from left to right: olive isolate (wild isolate), chrysanthemum isolate (wild isolate), olive isolate (subculturc from a variant colony sector), artichoke isolate (wild isolate), artichoke isolate (wild isolate), olive isolate (wild isolate). artichoke isolate (variant sector), olive isolate (variant sector). Profils Plectrophoritiques des proteines myckliennes totales des isolats de V . duhliue sur u n gel de polyacrylamide SDS a densite progressive (10-20?41).

resulting suspension was filtered through cheesecloth to remove mycelial fragments. After filtration the inoculum concentration was adjusted to 1 x 10" conidia ml-I. Herbaceous test plants were inoculated by the root-dip method (Matta et ul., 1980) and placed in a glasshouse at

Two-year-old olive plants, propagated as rooted cuttings, were used to test the susceptibility of olive cultivars to V. duhliue infection. Two methods of inoculation were used. In the first method the roots were dipped into the inoculum suspension before transplanting the plants into pots containing a steam-sterilized soil mixture (Cirulli & Montemurro, 1976). Control trees were dipped into tap water. After inoculation, the plants were placed into a growth chamber a t 22- 24°C under natural light with a 16-h photoperiod. Alternatively olive plants were inoculated by injecting a drop of the inoculum suspension into the stem through a cut made with a scalpel. Two inoculations were made on each plant on opposite sides of the stem, 5 cm above the soil level. Control trees were inoculated with sterile distilled water. After inoculation the plants were placed in a growth chamber a t 22-24°C and with a 16-h photoperiod. Alternatively they were transferred to an experimental field under natural environmental conditions. In the latter case symptoms were rated 7 months after inoculation on a 0 to 4 scale according to Tjamos et ul. (1 99 1).

20-26°C.

Resu Its

Pathogenicity tests on herbaceous hosts

Both V. duhliue isolates caused wilt in seedlings of cucumber (cv. Sprint), aubergine (cv. Violetta), capsicum (cv. Roco), tomato (cv. Super Marmande), and watermelon (cv. Crimson

Susceptibility to ver ticillium wilt 539

Table 1. Pathogenicity of two Verticillium dahliae isolates on herbaceous hosts (number of plants wilted after 5 weeks, out of 15 inoculated by the root-dip method) Pouvoir pathogene de deux isolats de V. duhliae sur des plantes herbacees (nombre de plantes fletries, apris 5 semaines, sur 15 inoculees par la methode de trempage des racines)

Host species

Verticillium duhliae isolates

Artichoke Olive isolate isolate

Cucumber (cv. Sprint) 15 5 Aubergine (cv. Violetta) 8 8 Pepper (cv. Roco) 11 5 Tomato (cv. Super Marmande) 11 5 Watermelon (cv. Crimson sweet) 15 13

Table 2. Mean verticillium wilt intensity a.b, and mean height in cm, on 2-year-old olive trees stem-inoculated with two isolates of Verticillium duhliae Intensite moyenne de verticilliose, et dimension moyenne en cm de plants d’olivier de 2 ans inocules au niveau de la tige avec deux isolats de V. duhliae

Verticillium duhliue isolates

Olive Artichoke Olive cultivar isolate isolate Control

Diseuse intensity 7 months after inoculation‘ Cassanese 1.7 E Nocellara del Belice 1.0 D Carolea 0.8 CD Cipressino 0.1 AB

Height in ern 4 months after inoculationd Cassanese 172 B Nocellara del Belice 132 B Carolea 153 A Cipressino 157 A

1.1 D 0.6 C 0.3 B 0.0 A

169 B 136 B 156 A 160 A

0.0 A 0.0 A 0.0 A 0.0 A

181 A I50 A 157 A 158 A

a Symptoms were rated on a 0 to 4 scale according to Tjamos et ul. (1991) Twenty trees of each cultivar per isolate were inoculated. Values followed by the same letter do not differ significantly according to Duncan’s multiple range test

Values followed by the same letter do not differ significantly according to Student’s t-test (P=O.Ol) . (P=O.Ol) .

Comparisons were made between inoculated trees and trees of the same cultivar used as controls.

Sweet). The isolate from artichoke was more virulent than the olive isolate (Table I). Both isolates induced severe stunting, leaf yellowing, and a black discoloration of the xylem in seedlings of turnip kale (Brassica oleracea var. gongylodes) cvs Express and Winner. None of the control seedlings showed symptoms. V. dahliae was recovered in pure culture on PDA from inoculated plants but not from control plants.

540 A . M . Pennisi et al.

Pathogenicity tests on olive

Cuttings from olive cvs Carolea, Cipressino, Coratina, Nocellara Etnea, Tonda Iblea, and Uovo di Piccione were inoculated with the V. dahliae isolate from olive, using the root-dip method. Twenty trees per cultivar were inoculated. Cvs Nocellara Etnea, Tonda Iblea, and Uovo di Piccione were most susceptible, with 100% of inoculated plants wilting within 70 days of inoculation. Cv. Carolea was the least susceptible (20% plants wilted) followed, in increasing order of susceptibility, by Cipressino and Coratina (40 and 60'K plants wilted, respectively).

In the next experiment, cuttings of the same cultivars were stem-inoculated with the V. dahliae isolate from olive. Once again cv. Carolea was the least susceptible: 20% of plants, out of 20 inoculated, wilted within 70 days of inoculation. Cvs Cipressino and Coratina also proved to be moderately susceptible (both with 25Y0 of plants wilted), while Nocellara Etnea, Tonda Iblea and Uovo di Piccione were very susceptible (100, 80, and 60'K1 of plants wilted, respectively).

In a third experiment, trees of cvs Carolea, Cassanese, Cipressino and Nocellara del Belice, propagated as rooted cuttings, were stem-inoculated either with the olive or the artichoke isolate of V. daliliae. Twenty trees were used per cultivar and per isolate, and 20 control trees. The trees were inoculated in late April and placed in the field. Symptoms were rated after 7 months (Table 2); however, a reduction of growth as a consequence of inoculation was observed before any symptoms of defoliation or wilt (Table 2). The cultivars which showed a significant growth reduction were those which subsequently proved to be the most susceptible on the basis of symptom severity, i.e. Cassanese and Nocellara del Belice. The isolate of V. dahliae from olive was more virulent than the artichoke isolate. Differences of susceptibility among olive cultivars were more evident in trees inoculated with the latter isolate. In contrast with the previous experiments, cv. Cipressino was less susceptible than Carolea, which, in turn, showed less severe symptoms than Cassanese and Nocellara del Belice. Control trees showed no symptoms. V. dahliae was recovered from all inoculated trees but not from control trees.

Discussion

The results of the pathogenicity tests are consistent with previous reports indicating that heterologous V. ciahliae isolates may infect olive (Schnathorst & Sibbett, 1971; Cirulli, 198 1). The present report of an isolate from artichoke capable of infecting olive extends the list of cultivated host species which could be a potential souree of V. dahliae inoculum when they are cultivated near or together with olive trees.

Cv. Carolea proved to be only moderately susceptible to V. dahliae infections compared with other olive cultivars commonly grown in Sicily and Southern Italy, such as Cassanese, Nocellara del Belice, Nocellara Etnea, and Tonda Iblea, which in contrast proved to be very susceptible. Coratina, a cultivar originating from Puglia, turned out to be susceptible to moderately susceptible. In previous tests made by others, this cultivar was tolerant to V. dahliae infections, i.e. it harboured the fungus showing only mild disease symptoms (Cirulli & Montemurro, 1976).

In ranking the susceptibility of olive cultivars to V. dahliae infections there was a good agreement between the results obtained using the root-dip or the wound-stem inoculation method. The latter seems more practical as it is not necessary to uproot the test plants and can be used to inoculate mature trees. The age of the plant can influence susceptibility or resistance to V. dahliae infections, thus pathogenicity tests on mature trees could reflect the actual field susceptibility of olive cultivars to verticillium wilt better than tests on young cuttings.

The high verticillium wilt incidence observed in the nursery near Milazzo is apparently related to the propagation system used to produce the trees. There is evidence to indicate that propagation of olive by rooted cuttings favours V. dahliae infections. In Italy production of olive cuttings is largely confined to nurseries located in Central Italy which use mist propagation systems. Infected cuttings produced in areas where verticillium wilt is widespread could rapidly

Susceptibility to verticillium wilt 54 1

spread the disease in olive-growing regions, such as Sicily and Calabria, where till now it has appeared only sporadically.

Evaluation de la sensibilite des cultivars d‘olivier a la verticilliose

Nous avons etudie la sensibilite a la verticilliose de divers cultivars commerciaux d’olivier. Deux isolats de Verticillium duhliue, provenant de l’olivier et de l’artichaut, ont kte uti1isi.s pour les tests. Des boutures racinees de 2 ans ont t t e inoculkes, soit par trempage, soit par injection dans la tige. Ces methodes ont donne des resultats comparables. Les cvs Carolea et Cipressino se sont rkvC1es moderement sensibles, alors que Cassanese, Nocellara del Belice, Nocellara Etnea, Tonda Iblea et Uovo di Piccione etaient tres sensibles. La reaction du cv. Coratina a fluctut de sensible a moyennement sensible.

O I \ e H K a n O ~ B e p X e H H O C T U MaCJlUH B e p T U q U n J I e 3 H O M y B U n T Y

B HaCTOSIIlefi pa60Te 6bina AaHa OUeHKa nOnBepXeHHOCTM pa3JIM’lHblX KOMMePqeCKMX COpTOB MaCnHH BepTblL(MnJle3HOMy BMJITY. Ann HCflblTaHMfi Ha ITaTOreHHOCTb 6b1m B3RTbl nBa M30nHTa

3apaXeHbl nw60 IIyTeM IIOrpyXeHMR KOpHefi, nw60 BHeCeHMeM MHOKyn5ITa B CTBOJI, npkiqeM B

JIHCb y M e p e € I H O B O C n p ~ l M M ~ ~ B b l M ~ l B TO BpeMH, KBK COpTa CaSSaneSe, Noccelara del Belice,

HOCTbH). nOBeneHlle COratina BapbMpOBanO MeXAy BOCflPMMMqMBbIM B yMepeHHOBOCIIpBMMqM- BblM.

Verticilliurn dahliae, nonyqetrribie c Macnm a apTmuoKoa. AeyxneTHne qepeHKA c KOPHRMM 6b1ns

odonx cnysarrx 6blnw nonyqesbr onaHaKoBbie pe3ynb~a~br. CopTa Carolea II Cipressino 0 ~ a 3 a -

Nocellara Etnea, Tonda Iblea M Uovo di Piccione o 6 n a n a n ~ spe3sbiqaA~o ebicoKoi4 syscramenb-

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