First report of Plasmodiophora brassicae in Lao People’s Democratic Republic

Cabbage production is promoted as a cash crop for small-land holders to support poverty alleviation on the Bolaven Plateau in Champasak Province, Lao People’s Democratic Republic (PDR). A stunted cabbage crop in Paksong was inspected due to a near total yield loss. Examination of the root systems revealed the presence of enlarged roots. Subsequently the disease was found in another round-head cabbage crop, and in a Chinese cabbage crop in a polyhouse. Air-dried clubroot specimens were forwarded to Australia where molecular methods confirmed the presence of Plasmodiophora brassicae. This is the first report of the obligate pathogen Plasmodiophora brassicae in the Lao PDR.

cabbages and other Brassica species in Europe, the UK and North America at that time (Woronin 1878).It is now distributed world-wide, causing losses of 10-15% annually (Dixon 2009).For example, in recent decades the disease has caused serious losses (up to 80%) to cabbage production in parts of Vietnam, notably the Da Lat area of Lam Dong province in the central highlands, and the Sapa area of Lao Cai province in the north-western mountainous region (Milham et al. 2020).However, clubroot disease had not been reported from neighbouring Lao People's Democratic Republic (PDR).
Cabbage production, predominantly round-head cabbages, has been promoted as a cash crop for small-land holders on the Bolaven Plateau in Champasak Province, since the early 2000s, to support poverty alleviation.The elevation of the cropping areas on the plateau varies from approximately 1100 to 1300 m ASL (Fig. 1) and the volcanic soils (ferrosols) and temperate climate provide ideal conditions for the production of temperate vegetable crops such as cabbages.
Members of the Champasak Agriculture and Forestry Office (PAFO), Australian Government Volunteers in plant pathology, and mentors supported by the Crawford Fund of Australia, have monitored cabbage crops on the Bolaven plateau for diseases and pests since 2009.In 2013, Rhizoctonia collar and root rot was detected and caused serious Plasmodiophora brassicae Woronin is an obligate biotrophic protist in the eukaryotic supergroup Rhizaria Cavalier-Smith (Schwelm et al. 2016).It was first described and shown to cause cabbage hernia (clubroot disease) in the classic publication by M. S. Woronin in 1878(Woronin 1878).The disease was a major cause of yield loss in losses at the seedling stage and stunting of older plants in cabbages and other Brassica crops on the plateau, and on the lowlands along the Mekong River near Pakse (Fig. 1) (Ireland et al. 2015).There was also evidence that soil fertility in cabbage fields on the plateau had declined with continuous cropping.However, clubroot disease caused by Plasmodiophora brassicae, had not been detected.
During annual surveillance for clubroot from 2014 to 2019, approximately 100 root systems were inspected after harvest in five to ten crops selected on an ad hoc basis in villages and on local roads along Highway 16E between Paksong and Thateng (Fig. 1).Crops were also checked pre-harvest for stunted plants, and wilting on hot days, key indicators of the disease, again on an ad hoc basis.Crops varied in area from approximately 0.2 ha to 5.0 ha across 27 villages in the Paksong district.More recently some cabbage crops have also been grown in large company-owned polyhouses.Awareness of clubroot disease as an exotic threat has been addressed in discussions with small-holder farmer in the field and at a workshop in 2014 small-holder farmers were asked to alert PAFO staff if the disease was detected.In June 2021, the first author was asked to inspect a stunted round-head cabbage (Brassica oleracea L. var.capitata) crop (3 ha) that was a total loss in Hauy Set Village near Paksong (15°13'07" N 106°18'17" E) (Fig. 2a and b).An inspection of the root systems revealed the presence of enlarged club shaped roots (Fig. 2b).Photographs of the crop and the root systems were forwarded to the Australian authors who confirmed that the symptoms were typical of clubroot disease.Clubroot specimens were then forwarded to the Plant Protection Centre in Vientiane for further confirmation.Subsequently the disease was found in another roundhead cabbage crop, and in a Chinese cabbage (Brassica rapa ssp.pekinensis) crop being grown in a polyhouse, both near Ka Taud Village, Paksong (Fig. 3a and b).
Air-dried clubroot specimens were forwarded under an Australian Quarantine and Inspection Service (AQIS) permit to Australia and vouchered at the Plant Pathology and Mycology Herbarium of the New South Wales Department of Primary Industries (NSW DPI) as DAR 85,816.A small segment of one clubroot specimen was forwarded to the Elizabeth Macarthur Agricultural Institute (EMAI) of NSW DPI for confirmation of the presence of Plasmodiophora brassicae using molecular methods.
For molecular identification, total nucleic acid was extracted from 250 mg of root tissue using the Ambion PureLink RNA kit (Invitrogen™, USA).Some modifications were made to the manufacturer's protocol; the tissue was ground over liquid nitrogen, resuspended in 0.5 mL lysis buffer and 1% v/v beta-ME, vortexed for 1 min, and then centrifuged ~ 5,000 x g for 5 min.Next, 0.4 mL of cleared lysate was transferred to a 1.5 mL tube with 0.2 mL absolute ethanol and mixed by pipetting before being transferred to a PureLink filter cartridge.The remainder of the protocol was as per manufacturer's instructions.The PCR was performed using primers TC1F (5'-GTG-GTCGAACTTCATTAAATTTGGGCTCTT-3')/TC1R (5'-TTCACCTACGGAACGTATATGTGCATGTGA-3') and TC1R (5'CTTTAGTTGTGTTTCGGCTAGGATG-GTTCG-3') (Cao et al. 2007).The amplification cycle consisted of an initial denaturation step of 94 °C for 2 min, followed by 45 cycles of 94 °C for 30 s, 65 °C for 45 s and 72 °C for 45 s, with a final extension at 72 °C for 10 min.
The PCR product was visualised on a 1.5% agarose gel to confirm the presence of the expected 548 bp amplicon.The amplicon was purified using the ISOLATE II PCR and Gel Kit (Bioline, United Kingdom) before being sent to the Australian Gene Research Facility (AGRF) (Sydney, Australia) for sequencing.The forward and reverse sequences were manually trimmed with reference to the chromatograms and then assembled using de novo assembly with default settings on Geneious Prime Version 2021.2.2.The concatenated sequence was compared to those of reference strains by BLASTn search analysis on GenBank (NCBI).
The BLASTn results confirmed DAR 85,816 had 100% sequence similarity to several Plasmodiophora brassicae accessions on GenBank.The sequence was deposited in GenBank under ON000243.1.
Due to the obligate nature of this pathogen, and its specialised host range restricting it to Brassica species, the authors agree that Koch's postulates is not necessary to confirm Plasmodiophora brassicae as the causal agent of the clubroot disease observed in Brassica spp. in Champasak Province, Lao PDR in 2021.The symptom observations and molecular data presented here constitute a first report of the pathogen and disease in the Lao PDR.
Clubroot disease represents a serious threat to the smallholder cabbage crops on the Bolaven plateau as the abundant resting spores of P. brassicae are persistent in soil for some years (Wallenhammar 1996;Zahr et al. 2021).Furthermore, it will be difficult to prevent the movement of infested soil Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. between small-holder farms via contaminated farm equipment, cattle, and footwear.Brassica weeds can also contribute to carryover of inoculum (Ren et al. 2016;Zamani-Noor et al. 2022), but few such weeds have been observed in the cabbage fields on the Bolaven plateau.However, there are a range of integrated disease management (IDM) strategies that could contribute to a reduction of inoculum levels in soil and hence a reduction in losses in yield.Woronin (1878) promoted crop rotation, the planting of disease-free seedlings, and the removal and burning of diseased plants, strategies which are still promoted today (Howard et al. 2010).In addition, resistant cultivars are available for some brassica crops, however, these may not be accessible in Lao PDR.It is now known that the clubroot disease is favoured by acid soil conditions so liming to about pH 7 has been promoted as a management strategy (Webster and Dixon 1991;Melland et al. 2020).More recently the pesticide fluazinam has been shown to reduce the impact of clubroot (Donald et al. 2001;Milham et al. 2020).The nature of the disease, and potential IDM strategies will be discussed with small-holder farmers on the Bolaven plateau through in-field meetings and village workshops as was done in the Sa Pa region of Vietnam where clubroot became a serious problem (Milham et al. 2020).These discussions will take into account that small-holder farmers are usually resource poor, and have limited knowledge of plant disease, issues considered in detail by Nelson et al. (2001).

References
Acknowledgements The authors gratefully acknowledge financial support from the Crawford Fund of Australia.The authors are also grateful for the support of staff of the Champasak Provincial

Fig. 1
Fig. 1 Map of Lao People's Democratic Republic showing main cities and closest large towns to surveyed cabbages (Thateng and Paksong); inset shows Lao PDR (grey) which is landlocked by five surrounding countries (yellow)

Fig. 2
Fig. 2 a) Stunted and wilting cabbage with club root; b) Cabbage with enlarged root with crop stunted and wilting from club root disease in background

Fig. 3
Fig. 3 a) Close-up of club-root infected round head cabbage; b) Close-up of club-root infected Chinese cabbage