Abstract
Rhizoctonia solani Kühn (teleomorph = Thanatephorus cucumeris (Frank) Donk) is one of the important soil-borne fungal pathogen, which infects tomato with typical symptoms of seedling damping-off and foot rot. During surveys (2014 and 2015 crop season) of nine tomato growing areas in Pothohar region of Pakistan, symptoms of foot rot were noted on approximately 33.4% of the plants observed at soil line level of the stem. Lesions on infected plant stems were irregular in shape, water-soaked, brown in colour manifesting sunken appearance. Fungal colonies isolated from stem portions of the diseased plants on malt extract agar medium were light grey to brown in colour with abundant mycelial growth and branched hyphae. A septum was always present in the branch of hyphae near the originating point with a slight constriction at the branch. No conidia or conidiophores were observed. All isolates were multinucleate when subjected to DAPI (4′,6-diamidino-2-phenylindole) stain. Based on morphological characteristics of fungal hyphae, isolates were identified as R. solani. Restriction analysis of PCR-amplified ribosomal DNA with four discriminant enzymes (MseI, AvaII, HincII, and MunI) and hyphal interactions with known tester strains confirmed these isolates belong to AG-3-PT (64.2%), AG-2-1 (14.2%), AG-2-2 (9.5%), AG-5 (7.1%) and AG-4-HGI (4.7%). AG-3-PT was widely distributed to major tomato growing areas while other groups were confined to distinct locations. Internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced which had 99–100% identity with the corresponding gene sequences of respective R. solani AGs. To confirm Koch’s postulates, four week old tomato plants were transplanted into 1.5 L plastic pots containing sterilized potting mixture i.e. sand: clay: farmyard manure, at the rate of 1:1:1. Soil inoculum containing 10 g of barley grains colonized with each isolate of R. solani for 14 days was mixed in the upper 2 cm layer of soil (Taheri and Tarighi, 2012). A set of uninoculated plants was used as a control. Ambient conditions were provided under the greenhouse. 21 days after inoculation, water-soaked greyish to brown lesions similar to the symptoms of the previous infection were observed on stem portions of all inoculated plants while control plants remained symptomless. Fungus re-isolated from infections was confirmed as R. solani by microscopic appearance of the hyphae. Present study is the first report of AG composition of R. solani infecting tomato in Pakistan which will be useful to breeding programs working on varietal evaluation.
Highlights
Solanum lycopersicum L., formerly known as Lycopersicon esculentum Mill., is one of the dominant vegetable crop worldwide that is generally cultivated in warm or tropical climate
Maximum mean disease incidence was observed in Islamabad (38.7%) followed by district Attock (36.3%), district Rawalpindi (34.9%) and district Chakwal (29.6%) while minimum mean disease incidence was recorded in district Jhelum (27.5%)
A total of 67 isolates of R. solani recovered on water agar (WA) medium started the hyphal growth from the second day of incubation
Summary
Solanum lycopersicum L., formerly known as Lycopersicon esculentum Mill., is one of the dominant vegetable crop worldwide that is generally cultivated in warm or tropical climate. AGs of R. solani are identified on the basis of hyphal anastomosis reactions, reproducibility of these interactions needs experiences, is a time consuming process and can be affected by factors including laboratory environment, nutritional conditions and genetic stability[14,16]. Foot rot of tomato caused by R. solani has already been reported from Pakistan AG group composition of R. solani responsible for this infection is not determined. The objective of the present study is to determine anastomosis groups (AGs) of R. solani causing foot rot of tomato using tester isolates and restriction analysis of ribosomal DNA
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