The health report - emerging pulse root diseases

Take home messages

• Pulse and canola crops can suffer from root diseases.
• Next generation sequencing technology and PREDICTA^®B tests are revealing multiple potentially important soilborne pathogens of pulse and oilseed crops in Australia; further work is being undertaken to determine which are the most important.
• In 2020, if you suspect soilborne disease issues in your pulse/canola crops, send samples to SARDI.

Background

International experience indicates that soilborne pathogens can be important constraints to production in pulse crops when cropping frequency increases (Gossen et al. 2016). In 2017, the loss of three chickpea crops to suspected Phytophthora root rot and a faba bean crop to Aphanomyces root rot, prompted the South Australian Grains Industry Trust (SAGIT) to fund a root disease survey of pulse and oilseed crops in South Australia (S218).

Phytophthora root rot, caused by Phytophthora medicaginis, is an important root disease of chickpeas in northern NSW. However, P. medicaginis was eliminated as the cause of loss of the three chickpea crops in South Australia (SA), using an existing PREDICTA^®B (Northern Region) test.

New diagnostic research technology being developed by the GRDC-SARDI bilateral investments; DAS1907-001BLX and DAS1802-011BLX was used to test DNA extracted from the diseased chickpea roots and identified Phytophthora megasperma as the likely cause. A PREDICTA^®B test was developed to support the survey.

In 2019, GRDC extended the survey nationally as part of DJP1907-002RMX. A panel of 23 tests was assembled to survey the pulse and oilseed root systems collected from across Australia (Table 1 and Table 2). DNA extracted from these samples was also tested using next generation sequencing (NGS), to detect pathogens for which no PREDICTA^®B-style test had been developed. The NGS data is still being examined.

Methods

Pulse root samples were sent to SARDI by growers and agronomists across SA. Excess soil was washed from the roots and any plant material above the basal stem was removed. Roots were processed through the PREDICTA^®B laboratory and DNA was extracted. The Pulse Research test panel was run on the extracted DNA to quantify targeted pulse pathogens in the samples.

DNA samples were also assessed using NGS to identify potentially important pathogens not detected by the Pulse Research test panel. Three Illumina^® MiSeq^® libraries were prepared using primer pairs that target the ITS1, ITS2 and elongation factor gene regions to aid identification of oomycetes (for example, Phytophthora) and fungal species (for example, Phoma and Fusarium species).

Where root samples showed distinctive/diagnostic symptoms, or where DNA tests indicated the presence of a potential pathogen, samples were plated on a variety of selective agar media in an attempt to culture the suspected pathogen(s).

Isolates are currently being tested for pathogenicity using the original host crop in a replicated controlled growth room bioassay (pot test). In short, 50ml tubes of sterile sand were planted with a seed of the host crop and inoculated with two millet seeds colonised by the cultured fungus. Plant roots were washed out approximately three weeks after sowing and disease was scored visually in comparison with an uninfected control. For isolates that appear pathogenic, a more representative bioassay will be performed, including other pulse species of interest. This will enable the pathogen’s host range to be determined.

Results and discussion

To date 400 samples have been processed from across all cropping regions in Australia, including 97 collected in 2018 from SA and western Victoria (Vic). Crops tested include chickpea, lentil, faba bean, field pea, lupin, canola, vetch, clover and lucerne.

Fifty-six isolates have been retained and sequenced using Sanger sequencing to identify isolates to species level. Of this collection, 20 Fusarium spp. isolates have undergone initial pathogenicity testing.

Pulse Research test panel

The results for the Pulse Research test panel are summarised in Table 1 and Table 2. Pratylenchus neglectus, Pythium clade F and Phoma pinodella (this test also detects Didymella pinodes) were all common.

P. neglectus (root lesion nematode) was detected at substantial levels in many crops including some that were considered to be poor hosts (for example, faba bean and field peas); presumably it does not multiply well in these crops even though it can invade the roots. Its effect on yield of pulses is not known.

P. pinodella has a broad host range amongst pulse crops and pasture legumes. It commonly causes foot rot in field pea and sub clover; it is also part of the pathogen complex causing black spot of field pea. Its effect as a pulse root pathogen is unknown.

Aphanomyces euteiches was found in 18% of samples in 2018 and 1% in 2019, all were from faba bean crops exhibiting moderate to severe root disease. In 2019, a test for Aphanomyces trifolii was added to the panel, with six samples (faba bean, lentil, vetch) found to be infected. The pathogen was particularly prevalent in vetch (27% of vetch samples infected). This pathogen is typically associated with sub-clover (O’Rourke et al. 2010). The effect of A. trifolii on lentil and vetch has not been described, while the effect on faba bean has only been briefly described (O’Rourke et al. 2010).

Rhizoctonia solani AG8 and AG2.1, Pythium clade I and Macrophomina phaseolina (charcoal rot) were also present at substantial levels. R. solani AG4, which can be a serious pathogen of pulses and other crops (Hwang et al. 2003), was detected in one sample in each year (chickpea and faba bean).

P. medicaginis was not detected in either year, probably due to drought in north Australia. Conditions were conducive for Phytophthora in the GRDC Southern and Western Regions of Australia and P. megasperma and P. clandestina were detected in SA and Western Australia (WA) (lentil and lupin). Both species are known to have a wide host range, however their importance in southern Australian pulse crops is yet to be quantified.

Table 1. Percentage of samples in which the stated pathogen was detected by a quantitative polymerase chain reaction (qPCR) in a survey of pulse roots in the South East region of SA and the Wimmera region of Victoria in 2018.

*Other crop types include vetch, canola and clover.

Table 2. Percentage of samples in which the stated pathogen was detected by a quantitative polymerase chain reaction (qPCR) in a survey of pulse roots from Australia in 2019.

Next Generation Sequencing (NGS)

DNA from each root sample was analysed with NGS and a broad range of fungal organisms were detected; some of which have been reported to cause root disease of pulses. Organisms were identified as pathogens of interest based on international research and observed symptoms in plant samples. Pathogens of interest identified in this survey are summarised as follows:

Phytophthora spp.

Sequence data identified several Phytophthora species present including P. megasperma/ crassamurra, P. trifolii and P. clandestina. All three species were detected in chickpea roots with symptoms of Phytophthora root rot in 2018 (i.e. year prior to sampling). P. megasperma/crassamurra was also found on faba bean and lucerne roots. These Phytophthora species could have been the pathogens responsible for crop failures in the chickpea paddocks from 2017 and crop and root symptoms in 2018. These samples were negative for P. medicaginis using the PREDICTA^®B test. The potential of P. megasperma/crassamurra to also infect faba bean roots could have implications for the South East region and requires further investigation to confirm and quantify its extent and severity.

Australian research in the GRDC Northern Region on Phytophthora root rot (P. medicaginis) is currently the best reference point for the impacts of this disease in chickpea as the impacts in the GRDC Southern Region are currently unknown. Further research in the GRDC Southern Region is required to determine the severity of impact of P. megasperma/crassumurra and how it compares to Phytophthora root rot in the Northern Region where it was estimated to cost chickpea growers up to $8.2 million annually (Murray and Brennan, 2012).

The only chemical option available for Phytophthora root rot in the GRDC Northern Region is metalaxyl-based seed dressings, which can provide six to eight weeks protection post seeding (Moore et al. 2011). Currently, the best non-chemical options for growers to manage Phytophthora root rot are to use wide rotations and grow varieties that are moderately resistant (Amalraj et al. 2018).

Thielaviopsis basicola

Thielaviopsis basicola sequences were detected on diseased chickpea roots grown in soil from near Naracoorte and from a diseased lupin root system from Coomandook in 2019. T. basicola causes black root rot and has a very broad host range including pulses, vegetables and cotton. Internationally, chickpea and lentil have been identified as susceptible to T. basicola, and disease has been found in numerous cropping regions (Bowden et al. 1985, Abbas et al. 2007, Bhatti et al. 1992). A test for T. basicola has now been added to the PREDICTA^®B Pulse Research test panel, with one lupin sample from WA returning a positive detection in 2019.

Fusarium spp.

Globally, Fusarium spp. feature frequently in research on pulse root diseases (for example, Gossen et al. 2016, Li et al. 2017, Wong et al. 1985, Banniza et al. 2015). Species reported in the literature and tentatively identified as detected by the survey, include F. solani, F. redolens, F. oxysporum, F. equiseti, F. avenaceum and F. acuminatum. Internationally, research groups are currently investigating the role of these species as potentially important components of disease complexes with A. eutiches and Phytophthora spp. (Banniza, 2016).

In North America and Canada, F. redolens is considered to be an important component of pulse root disease complexes. Following confirmation of the presence of F. redolens in Australia, SARDI developed a PREDICTA^®B style test for this species to assist with the survey. Tests for other Fusarium spp. may follow, depending on results of pathogenicity experiments.

There are constraints on the resolution of the NGS. The Illumina^® MiSeq^® sequences cannot differentiate Fusarium species to forma specialis. This limits our current ability to identify some of the most important root pathogens of chickpea (F. oxysporum f. sp. ciceris) and lentil (f. sp. lentis). Both however, are not currently known to occur in Australia (Cunnington et al. 2016, Pouralibaba et al. 2016).

Further investigation is needed to determine which, if any, of the above species play an important role in causing pulse root disease in Australia.

Pathogenicity testing

Screening of isolates of potential pathogens extracted from root samples was undertaken using a bioassay in a controlled growth room. Preliminary results are presented in Table 3. The results indicate that isolates BC10287a, BC10294(2), 10225(3), 10225(4), 10286(2) and 10286(9) are pathogenic and can cause considerable root damage. These isolates have been tentatively identified as belonging to a range of Fusarium (and Cylindrocarpon/Dactylonectria) species. Other isolates tested in this screening that were identified as the same species, were not pathogenic, or only weakly pathogenic. This confirms that there is likely to be considerable variation in pathogenicity; both between and within species of Fusarium and related genera.

A single isolate of Phoma pinodella (BLBG1) included in this test was moderately pathogenic. Isolates that rated moderately to strongly pathogenic in the initial screen will be included in a more extensive bioassay, while new isolates of many other genera including Phoma, Pythium, Phytophthora will also be tested in bioassays.

Table 3. Disease scores and recovery of pathogen from inoculated plant material from 20 fungal isolates from pulse roots surveyed in South Australia in 2018-19.

Conclusion

While this research is in the ‘problem definition’ phase, some patterns are beginning to emerge. Phytophthora and Aphanomyces have been associated with crop failures in the high rainfall zones, but their occurrence seems to be sporadic. Other potential root pathogens such as Fusarium, Pythium and Phoma are much more common, within and across regions. Their effect on yield needs to be further investigated however many species of these genera are known to be pathogenic. These potential pathogens being widespread suggests they have greater potential for impact across the industry.

It is likely that pulse root diseases are contributing to poor water use efficiency and unexpected yield losses, and the risk is likely to increase with increased frequency of pulses in cropping sequences. Legume pastures may also be a significant source of infection.

Research to evaluate the impact of pulse root diseases and management options are expected to commence in 2020. In the meantime, SARDI will continue to survey pulse crops in SA under the SAGIT project S218 in 2020, and nationally under the GRDC project DJP1907-002RMX. Consultants and growers are encouraged to monitor their pulse crops and submit plant samples from poor performing areas that previously may have been attributed to waterlogging or other environmental stressors.

If you are interested in assisting with the survey, please contact Blake Gontar for sample kits.

Acknowledgements

The research undertaken as part of this project is made possible by the significant contributions of consultants and growers through sample contribution and the support of GRDC and SAGIT (project S218); the authors would like to thank them for their continued support. From 2019 onward, this survey is being conducted in collaboration with researchers from WA, Queensland, New South Wales and Vic.

References

Abbas, S.Q., Niaz, M. and Ghaffar, A., 2007. Thielaviopsis basicola: a potential threat to agriculture and forestry in Pakistan. Pakistan Journal of Botany, 39(3), pp.985-990.

Amalraj, A., Taylor, J., Bithell, S., Li, Y., Moore, K., Hobson, K. and Sutton, T., 2018. Mapping resistance to Phytophthora root rot identifies independent loci from cultivated (Cicer arietinum L.) and wild (Cicer echinospermum PH Davis) chickpea. Theoretical and Applied Genetics, pp.1-17.

Banniza, S., Phelps, S. and Doggen-Bouchard, F., 2015. What in the soil is going on with prairie field pea production?

Banniza, S., 2016. Could someone please drain my soils? I want to grow pulses.

Bhatti, M.A. and Kraft, J.M., 1992. Reaction of selected chickpea lines to Fusarium and Thielaviopsis root rots. Plant disease, 76(1), pp.54-56.

Bowden, R.L., Wiese, M.V., Crock, J.E. and Auld, D.L., 1985. Root rot of chickpeas and lentils caused by Thielaviopsis basicola. Plant disease, 69(12), pp.1089-1091.

Cunnington, J, Lindbeck, K and Jones, RH, 2016. National Diagnostic Protocol for Fusarium oxysporum f. sp. ciceris – NDP36 V1. (Eds. Subcommittee on Plant Health Diagnostics).

Gossen, B.D., Conner, R.L., Chang, K.F., Pasche, J.S., McLaren, D.L., Henriquez, M.A., Chatterton, S. and Hwang, S.F., 2016. Identifying and managing root rot of pulses on the northern great plains. Plant Disease, 100(10), pp.1965-1978.

Hwang, S.F., Gossen, B.D., Chang, K.F., Turnbull, G.D., Howard, R.J. and Blade, S.F., 2003. Etiology, impact and control of rhizoctonia seedling blight and root rot of chickpea on the Canadian prairies. Canadian Journal of Plant Science, 83(4), pp.959-967.

Li, Y.G., Zhang, S.Q., Sun, L.P., Li, S. and Ji, P., 2017. First Report of Root Rot of Cowpea Caused by Fusarium equiseti in Georgia in the United States. Plant Disease, 101(9), pp.1674-1674.

Moore, K., Ryley, M., Schwinghamer, M., Cumming, G. and Jenkins, L., 2011. Chickpea: Phytophthora root rot management. Pulse Australia Disease Management Series PA2011, 11.

Murray GM, Brennan JP (2012). The current and potential costs from diseases of pulse crops in Australia: GRDC Research Code: CER00002. GRDC, Barton ACT.

O’Rourke, T.A., Ryan, M.H., Li, H., Ma, X., Sivasithamparam, K.,  Fatehi, J. and Barbetti, M.J. (2010) taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii causing root rot of subterranean clover (Trifolium subterraneum) in Western Australia. Crop & Pasture Science, 61, pp.708–720.

Pouralibaba, H.R., Rubiales, D. and Fondevilla, S., 2016. Identification of pathotypes in Fusarium oxysporum f. sp. lentis. European Journal of Plant Pathology, 144(3), pp.539-549.

Wong, D.H., Barbetti, M.J. and Sivasithamparam, K., 1985. Fungi associated with root rot of subterranean clover in Western Australia. Australian Journal of Exp Agriculture, 25(3), pp.574-579.

Contact details

Blake Gontar
SARDI Soil Biology and Molecular Diagnostics
Plant Research Centre, Gate 2B Hartley Grove, Urrbrae, SA, 5064
0430 597 811
blake.gontar@sa.gov.au