1. New host records for Verticillium tricorpus, Corynespora cassiicola, Aphelenchoides fragariae and Helicotylenchus dihystera, associated with dieback and leaf blight of hydroponically grown sweet basil (Ocimum basilicum) in Victoria, Australia
Aldaoud R, de Alwis S, Nambiar L, Salib S, Dinh SQ, Holmes RJ, Edwards J
Crop Health Services, Agriculture Victoria, AgriBio Centre, 5 Ring Road, La Trobe University, Bundoora, VIC 3083
INTRODUCTION
Crop Health Services (CHS) is a business within Agriculture Victoria that provides fee-for-service plant diagnostics for clients, as well as providing diagnostic capability to support Biosecurity Branch, the regulatory and compliance arm of Agriculture Victoria.
In February 2012, hydroponically grown sweet basil (Ocimum basilicum) plants were submitted by a commercial client to CHS for diagnosis of possible causes of brown necrotic patches on leaves (Figure 1, red arrows) and stems (Figure 1, white arrows). The grower had suspected Botrytis infection, but fungicide applications were ineffective.
FUNGAL ISOLATION AND IDENTIFICATION
Fungal isolation was undertaken aseptically, by first surface-sterilizing specimens taken from the affected tissues in solution of 0.5% NaOCl for two minutes, blotting dry on sterile tissue paper, and plating small pieces of infected tissue onto tetracycline-amended potato dextrose agar (PDAT), water agar (WA) and V8-juice agar plates, with seven pieces per plate. A Corynespora–like fungus (Figure 2, left) was frequently recovered from the necrotic external stem tissues. A Verticillium–like fungus (Figure 2, middle and right) was occasionally recovered from brown-discoloured vascular stem tissues. Both fungi were further subcultured on PDAT to generate clean cultures prior to molecular identification.
DNA was extracted from mycelium of pure cultures with Qiagen DNeasy Plant Mini Kits following the manufacturer's instructions. PCRs were performed to amplify the fungal ITS regions using the primers ITS4 and ITS5. Products were cleaned with a Qiagen QIAquick PCR purification kit and sequenced using Applied Biosystems BigDye technology. Sequences were corrected using DNA Baser V4 software and were identified using Blast searches of reference sequences on GenBank. The fungal isolates were closest matches to V. tricorpus (99% identity, 100% coverage) and C. cassiicola (100% identity, 100% coverage), respectively.
NEMATODE ISOLATION AND IDENTIFICATION
Nematode extractions were performed from the necrotic leaf patches and from the potting medium. Aphelenchoides fragariae (foliar nematode) was isolated from the leaves (Figure 4) and Helicotylenchus dihystera (spiral nematode, Figure 5) was present in very large numbers (21,700 nematodes/200 mL). Nematode identifications were confirmed by comparing key morphological characters with those of type specimens held in the VPRI reference collection.
OUTCOMES
This is the first time these pathogens (C. cassiicola, V. tricorpus, A. fragariae and H. dihystera) have been associated with sweet basil in Victoria. As required by legislation, these new records of pests and diseases were reported at the time to the Victorian Chief Plant Health Manager.  
We were unable to find a record of V. tricorpus on basil, although it has been recorded in NSW on datura and sesame.
Corynespora cassiicola has been previously reported on basil in Queensland, but this was a first report on basil for Victoria. This fungus has a wide host range and there are 113 records on the Australian Plant Pest Database.
Aphelenchoides fragariae and Helicotylenchus dihystera were both new host records for Australia, although they are present here on other hosts.
Sweet basil is grown in Victoria in protected cropping systems and the presence of all these pathogens on a single crop is of concern. This crop was one year old and the source of the pathogens was not able to be determined. The grower had suspected Botrytis to be the culprit, but after receiving the results of the CHS diagnosis, the crop was destroyed. This emphasises the importance of adhering to strict hygiene protocols for prevention of diseases in these closed systems.
Figure 1. Symptoms on sweet basil stems (left) and leaves (right)
Figure 2. Corynespora cassiicola conidia, scale bar = 25 µm
Figure 3. Verticillium tricorpus conidiophore and conidia
(left, scale bar = 10 µm) and microsclerotia (right, scale bar = 25 µm)
Figure 4. An adult Aphelenchoides fragariae nematode emerging from infested leaf tissue, scale bar = 20 µm
Figure 5. An adult Helicotylenchus dihystera nematode