First Report of Armillaria Root Rot Caused by Armillaria mellea Infecting Carrizo Citrange and Sour Orange Rootstocks in Turkey.

Abstract

Citrus rootstocks, Carrizo citrange (Citrus sinensis [L.] Osb. × Poncirus trifoliata [L.] Raf.) and sour orange (C. aurantium L.) grown in containers filled with 5 liters of potting mix of 40% peat and 60% volcanic tuff declined in a 0.2-ha commercial nursery in Adana, Turkey, between 2004 and 2007. Seedlings with symptoms of root rot were found with an average disease incidence of 20% among 1,000 Carrizo citrange seedlings and 10% among 15,000 sour orange seedlings. The potting mixture preparation unit was located next to an oak tree (Quercus sp.) showing symptoms of Armillaria root rot. Six- to 12-month-old seedlings of both rootstocks were stunted and the crowns were necrotic with the presence of white mycelium. Mycelial fans were observed beneath the bark of infected roots and they expanded into the crown. The root systems and nearby potting mix contained rhizomorphs. Thus, Armillaria spp. was suspected as a possible causal agent. Three diseased crowns and three rhizomorphs were surface-sterilized with 1% NaClO for 1 min and cultured on benomyl-dichloran-streptomycin containing selective medium (3) at 25°C in the dark for 1 week. Six isolates transferred to 1.5% malt extract agar at 33°C in the dark for 7 weeks consistently yielded abundant aerial hyphae and mean diameter growth range was 4 to 21 mm and the mycelium margin was regular (1). To confirm pathogen identity, total DNA was extracted using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., CA) directly from 7-day-old cultures grown in potato dextrose broth (PDB). The ribosomal DNA internal transcribed spacer (ITS) region was amplified by PCR using the primer pair ITS1 and ITS4 (5) and sequenced. The sequences were 99% identical to that of Armillaria mellea isolates from Japan (AB510880) and China (KF032535). This confirmed the identity of the causal agent as A. mellea (Vahl.) P. Kumm. Ten 3-month-old seedlings of Carrizo citrange and sour orange were transplanted into steam-sterilized potting mix and inoculated with wood pieces of oak (Quercus sp.) colonized by the fungus (two pieces for each container) (2). The oak wood pieces were sterilized prior to the colonization by the pathogen. Plants were maintained in a greenhouse (23 to 25°C) until symptoms appeared. Ten non-inoculated seedlings from each rootstock served as controls and were maintained in the same environment. After 4 months, the crowns of the seedlings developed necrotic areas and root systems contained rhizomorphs on all inoculated seedlings and fungus was re-isolated from crowns and rhizomorphs. All control plants remained disease-free and no fungus was re-isolated. A. mellea was reported to infect citrus rootstocks in Spain in 1999 (4). To our knowledge, this is the first report of Armillaria root rot caused by A. mellea infecting Carrizo citrange and sour orange rootstocks in Turkey. This indicates that citrus rootstocks could be at risk for infection and sterilization of the potting mix and good sanitation practices in nurseries are very important.