ISOLATION AND IDENTIFICATION OF THE CAUSAL AGENTS OF ROOT AND CROWN ROT OF PISTACHIO TREES IN THE SISTAN AND BALUCHISTAN PROVINCES

Abstract

Pistachio is the most important commercial product in Iran and root and crown rot (gummosis) is the most serious diseases of this crop. Causal agents of the disease were unknown in Sistan and Baluchistan province where 5000 ha are under cultivation of pistachio. During 2002-2003, thirty pistachio orchards including pistachio trees of different ages were inspected throughout different seasons. Eighteen Phytophthora isolates were isolated using citrus leaves pieces as baits on water-saturated soils collected from around the infected trees and cultured on PARPH medium. Some isolates were isolated culturing the surface disinfected tissues of root and crown of infected trees directly on PARPH as the semi-selective medium too. Two groups of isolates were obtained. Based on the morphological and physiological characteristics of the Sistan and Bluchistan pistachio isolates, the first group of isolates was identified as Phytophthora pistaciae sp. nov. Mirabolfathy and the second group identified as Phytophthora nocotianae Bred de Haan. The two Phytophthora species were found pathogens using unripe pear and apple fruit inoculation method, detached pistachio twigs inoculation method and inoculated vermiculite method to infect the pistachio scions through the root area and artificial inoculation of the crown area with a plug of cultured isolate medium. This is the first report of incidence of pistachio tree gummosis and its causal agents from Sistan and Baluchistan province. INTRODUCTION One of the most limitative agents of pistachio cultivation in Iran is root and crown rot disease (gummosis) caused by Phytophthora spp. The causal agents of the disease in Kerman province, which is the biggest area in pistachio cultivation and production, are P. pistaciae, P. melonis, P. citrophthora, P. cryptogea and P. nicotianae, and in other regions such as Yazd, Qazvin, Semnan and Fars provinces, some of theses species of Phythophtora were reported. A few reports about pistachio gummosis agents are available in the other countries. P. citrophthora, P. citricola and P. nicotianae were reported from Greek orchards (Koyeas1973) and P. capsici and P. cryptogea were identified as stem and trunk dieback causal agents in the USA (MacDonald et al. 1992). Pistachio orchards are young in Sistan and Baluchistan province. The aim of this research was diagnosis of the causal agents of a disease unknown in this area. MATERIAL AND METHODS During 2002-2003, the pistachio orchards throughout this area were surveyed to find the causal agents of the disease. Thirty pistachio orchards including pistachio trees of different ages were inspected in Zahedan and Khash regions throughout different seasons. The soil around the root and the crown of infected trees with symptoms of gum oozing through the crown, leaf chlorosis, and decreasing of leaf covering were collected. Tissues of infected trees with symptoms of root and crown rot were also sampled. Isolates were isolated in three methods: isolation by baiting techniques with usage of apple fruit, unripe pear fruit and citrus leaf disks as the baits on water sutured soils collected from the infected trees; isolation by culturing the surface disinfected tissues of root and crown of infected trees directly; and culturing the surface tissues after washing thoroughly in 647 Proc. IV IS on Pistachios and Almonds Eds.: A. Javanshah et al. Acta Hort. 726, ISHS 2006 running water for several hours and without disinfections on CMA-PARPH as the semi selective medium. Disinfectants were % 6 H2O2 solutions and % 1 sodium hypochlorite. Isolates were purified on WA (water agar) medium after growth with hyphal tip method. Sporangia resulted when putting some pieces of young mycelium that grew on HBA (Haricot Bean Agar) medium in ordinary water, distilled water or soil extract. For oospore formation, LBA (Lima Bean Agar) medium was used. Homothallic oospores were formed when placing some patches of young colony margins on LBA medium. Isolates were stored on CMA medium in slant tubes on 15°C. Temperature effect was studied by means of daily radial growth measurement of isolates on CMA medium on 10, 15, 20, 25, 27.5, 30, 32.5, 35, 37.5 and 40°C. Pathogenicity tests were done by inoculation method on apple fruit, unripe pear fruit, detached pistachio twigs (Kalleh-Ghoochi cultivars), infested vermiculate pistachio scions (Sarakhs cultivars) and artificial inoculation of the crown area with a plug of cultured isolate medium on nine months scions. RESULTS AND DISCUSSION Eighteen Phytophthora isolates were isolated. Two groups of isolates were obtained. The colony morphology of twelve isolates as the first group was radial and regular on PDA, sporangia were formed in aquatic media only as non-papillate and spherical and ovoid. The average dimensions of sporangia were 30.28 × 38.96 μm with length breadth ratio 1.29:1, sporangia formed by external or internal proliferation (nesting). All of the above isolates were homothallic, antheridia attached to oogonia as paragynus mainly and amphigenus seldom, the average dimensions of oogonia, antheridia and oospores were 33.02, 8.85×10.84, 27.25 μm respectively. The minimum, optimum and maximum growth temperature for growth of above isolates were 15, 27.5 and 35°C, respectively, the daily radial growth rate of the isolates at optimum temperature was 1.95 mm. The second group involved six isolates, colony morphology of these isolates on PDA were arachnoids, sporangia were formed in the agar media as ovoid or spherical shapes with a prominent papilla with dimension of 24.96×32.47 μm (average) and theirlength breadth ratio was 1.31:1, sporangia formed singly or in a loose sympodial long sporangiophore. These groups of isolates were heterothallic and, chlamydospores were formed abundantly. The minimum, optimum and maximum temperature for growth of these isolates were 15,25 and 37.5,°C respectively. The daily radial growth at the optimum temperature was 2.15 mm. Based on the morphological and physiological characteristics of Sistan and Bluchestan pistachio isolates the first group of isolates was identified as Phytophthora pistaciae sp. nov. Mirabolfathy and the second group was identified as Phytophthora nicotianae Bred de Haan. Phythophthora species isolates were found pathogens using unripe pear and apple fruit inoculation method, detached pistachio twigs (Kaleh-Khoochi cultivar) inoculation method and inoculated vermiculite method to infect the pistachio scions (Sarakhs cultivar) through the root area and artificial inoculation of the crown area with a plug of cultured isolate medium. This is the first report of incidence of pistachio tree gummosis and its causal agents from Sistan and Baluchistan province. CONCLUSIONS The results from surveys of infested orchards of two different regions, Sistan and Baluchistan, with the necessary tests for identification of isolates, has lead to detection of twelve isolates of P. pistacia and six isolates of P. nicotianae. Therefore, the main agent of gummosis in Sistan and Baluchistan provinces is P. pistaciae (66.6% of isolates) and P. nicotianae is in the second rank(33.3% of isolates). This result corresponds to the conclusion of a disease investigation in Kerman province (Mirabolfathy et al. 1987 and Ashkan et al. 1995).