Materials and methods
After assessing standard operating practices at each nursery, a strategy was designed to sample potential contamination sites. These included:
- soil from dirt roads around tunnels and from walkways inside tunnels/netting structures
- unused potting media (from surface and deeper in the pile)
- potting media in use
- irrigation water sampled after flocculation, filtration or sterilisation
- surfaces sampled with sterile cotton swabs: wheelbarrows, tractor wheels, filled and empty seedling trays, worker boots and gloves, cement benches, shade netting, footbaths, and moss or plant debris in or around pots.
Soil samples were baited with citrus leaf discs on flooded slurries, then plated onto selective media. Putative Phytophthora isolates were purified, morphologically identified, and identities confirmed by Polymerase Chain Reaction (PCR) and sequencing of two Internal Transcribed Spacer regions. DNA was extracted directly from swab samples and subsequently screened with ITS primers to detect Phytophthora gDNA.
Results and discussion
All nurseries surveyed tested positive for Phytophthora inoculum in at least one sampled site. Notably, no oomycete-like organisms were isolated from any of the 110 water samples, confirming that the chlorination, flocculation, and filtration systems used in these nurseries are effective at eradicating oomycete species.
Phytophthora was recovered from soil on dirt roads surrounding tunnels (average 44% of samples positive across nurseries) and from tunnel walkways (41% positive). Inoculum was also present in potting media before use (17%), and in use (24%) at more than half of the nurseries sampled, despite nursery managers considering these media to be sterile due to composting, on-site treatment, or certified purchase.
Swabbing revealed even more contamination points. Phytophthora DNA was frequently amplified from:
- tractor wheels and wheelbarrows (movement of soil between areas)
- boots and gloves
- cement benches used to raise pots off the ground
- moss and plant debris in or around pots
- shade netting structures
- filled seedling trays and discarded used trays
- footbaths (even where disinfectant was used).
These results show that Phytophthora survives and spreads at multiple steps in the nursery. Equipment, vehicles, worker clothing and footwear, benches, trays and even mossy debris act as bridges that carry inoculum from contaminated soil or old media into fresh potting mix and onto healthy roots.
Why does the pathogen keep coming back? Poor drainage and runoff allow build-up near production areas. Surrounding soil likely re-contaminates treated media over time. Composting temperatures, or durations in some cases, appear insufficient to kill resilient Phytophthora propagules. Disinfectant footbaths and vehicle dips lose efficacy if not refreshed often enough. High plant density, frequent irrigation and warm conditions inside tunnels create an ideal environment for any surviving inoculum to multiply. The good news is that the tested samples of treated irrigation water were free from oomycetes, highlighting the value of the water-treatment and in-line spore trapping protocols already in place.
It is important to note that this data is merely a snapshot of the situation in nurseries. Nonetheless, the results highlight several practical steps for disease management, all of which are outlined in the CIS nursery management guidelines:
- Test potting media batches (new and in use) regularly for the presence of Phytophthora.
- Improve composting: ensure high enough temperatures and turning for long enough to eliminate oospores and chlamydospores.
- Clean and sanitise wheelbarrows, trays, benches, tractor wheels and shade nets more thoroughly and regularly.
- Replace footbath and dip solutions regularly to maintain effectiveness.
- Minimise standing water, excessive wetness, and runoff around potting areas.
- Keep walkways and surrounding roads free of plant debris and avoid bringing soil into tunnels on boots or tyres.
- Consider routine testing of high-risk surfaces with swabs or baiting to find hidden reservoirs early.
The results from this survey emphasise that successful Phytophthora management in citrus nurseries requires a systems approach – not just one or two control measures.
Acknowledgements:
We thank the nursery owners and staff who allowed sampling on their premises, and Christopher Liakos for valuable laboratory support.
Citrus Research International Jacquie E. van der Waals* and Jan M. van Niekerk
Department of Plant and Soil Sciences, University of Pretoria Jacquie E. van der Waals*, Jason McNeil and Khumbuzile N. Bophela
Department of Biochemistry, Genetics and Microbiology, University of Pretoria Teresa A. Coutinho
Department of Plant Pathology, Stellenbosch University Jan M. van Niekerk
*Corresponding author: jacquie@cri.co.za
