A new DNA tool to detect chickpea Phytophthora in paddocks

Take home message 

• Knowledge of Phytophthora medicaginis (Pm) DNA concentration in soil can assist Phytophthora root rot (PRR) management
• In a field trial, treatments with 0 and 100 oospores/plant) resulted in low Pm DNA concentrations and had significantly less disease and significantly higher yields than treatments with higher oospore concentrations
• Similar PRR disease levels and yield losses came from medium to high Pm soil inoculum concentrations for the Pm susceptible variety Sonali
• The Pm DNA test is capable of identifying Pm in soil samples from growers paddocks
• Pm DNA results and Pm isolation results agreed for most paddock samples with 82% of positive and 97% of negative (97%) being consistent. However, results for three samples indicate that further work is required to address some issues including subsampling effects

Note: the SARDI test for Phytophthora medicaginis is under development and is not yet available commercially

Phytophthora medicaginis detection in soil

Phytophthora medicaginis (Pm), the cause of Phytophthora root rot (PRR) disease of chickpea is endemic and widespread in southern QLD and northern NSW.  The pathogen carries over from season to season on infected chickpea volunteers, lucerne, native medics and as resistant structures (oospores) in the soil. 

A PreDicta B^® soil DNA test has been developed by the South Australian Research and Development Institute (SARDI) GRDC project DAS00137 to quantify the amount of Pm DNA in soil samples and to provide a measure of the amount of Pm inoculum (infected root tissue and oospores) in paddocks from which those samples were collected.  Studies are currently underway to assess the capability of this Pm DNA test to:

1. Quantify Pm inoculum in soil from commercial paddocks
2. Predict the risk of PRR disease and potential yield losses in chickpea

Pm inoculum level, PRR disease and yield

It would be useful if the DNA levels detected by the Pm test could predict PRR disease level and potential associated losses.  For example, would paddocks with nil, low and high Pm inoculum level respectively have nil, low and high PRR disease and yield losses?

This was the aim of a 2014 field trial at the NSW DPI Tamworth Agricultural Institute using the highly PRR susceptible chickpea variety, Sonali.  On 3 July 2014, a range of Pm inoculum levels was established by applying, at sowing, different rates of oospores in-furrow. On 4 Aug, when plants were at the 2 node stage, ten soil samples (150 mm depth cores) per plot were pooled and analysed for soil Pm DNA concentration by SARDI.  During the season PRR disease assessments and normalised difference vegetation index (NVDI) measurements were made and grain yields determined.  The trial was also sampled mid-season (4 August) and end-season (19 December) for Pm DNA quantification (data not available at time of writing).

The soil Pm DNA results differed significantly among the oospore treatments but also indicated that some Pm was already present at the site (Table1).  Background concentration effects could have contributed to the higher than expected value for the 100 oospores/plant treatment.  The season in Tamworth was drier than usual but following 39mm of rain from 18-20 August, some PRR symptoms (wilting, chlorosis) were observed, and in mid-September during a period (15-22 Sept.) of hot dry winds and high evaporative demand (≥ 5mm/day) many plots rapidly showed severe PRR disease symptoms.

In mid-September indirect biomass measurement by the reflectance based NVDI showed significant declines in NVDI values with increasing number of oospores/plant treatment.  By the end of September the 0 oospores/plant had less diseased plants than the 100 oospores/plant treatment, which itself had less diseased plants than the treatments with higher inoculum levels (500 to 4000 oospores/plant treatments). The percentage of diseased plants did not differ between the 500 to4000 oospores/plant treatments. The ranking of the 23 September disease assessment reflected the final grain yields for this trial, with the 0 and 100 oospores/plant treatments each having the highest and second highest yields respectively, and the yields of the 500-4000 oospores/plant treatments not differing.

Table 1. Oospore level, soil Pm DNA concentration, PRR assessment and yield in 2014 Pm inoculum level trial at Tamworth NSW (SED=Standard error of difference between means)

*100% values excluded from analyses

There were relatively weak correlations between the post-sow soil Pm DNA concentrations and PRR disease (r = 0.45) and chickpea yields (r = - 0.39), although the percentage of diseased plants was a strong predictor of grain yield (r = - 0.96).

Pm can reproduce rapidly and cause new infections over a relatively short period (Ristaino et al. 1993).  This may explain how the 500 to 4000 oospores/plant treatments had very similar PRR disease symptoms measurement values at 23 September, which then led to similar grain yields.  These results indicate that for a susceptible variety like Sonali, PRR disease can build up to high levels under conducive conditions and cause considerable yield losses despite differences in initial Pm inoculum levels. 

Although the relationships between Pm DNA concentrations and PRR disease and chickpea yields were relatively weak, the trial showed low Pm inoculum levels (0 and 100 oospores/plant) had significantly less disease and significantly higher yields than treatments with higher oospore concentrations. 

These initial results are encouraging as they suggest that significantly lower PRR disease and higher yields occur at low Pm DNA concentrations with a highly susceptible variety. The test may be suitable at identifying low Pm inoculum sites where chickpea varieties with better Pm resistance (such as Yorker) may be grown with less impact of PRR on yield.  The test may also be useful at identifying nil from low Pm sites, however, trial sites with nil Pm will need to be identified to fully assess this aspect.

Pm DNA detection in soil samples from commercial paddocks

We evaluated the ability of the Pm DNA test to detect Pm in soil samples from growers paddocks. 

Over the winter-spring period of 2013 soil samples were collected from fields in northern NSW and Southern QLD.  All paddocks included chickpeas in the rotation but not all had chickpeas in 2013.There were eight sample sites per paddocks, one near each corner and one near the midpoint of each side.  At each of the eight sample sites, a W collection pattern was walked towards the centre of the paddocks and 10 soil cores (150mm depth PreDicta B^® soil corer) collected every 20 – 25 paces along the sample path (total distance 200 – 250m per sample site).  If soil conditions prevented coring (ie too dry), a trowel (15cm long by 6cm wide tapering to 2cm) was used. The same W sample collection pattern as for the coring method was walked.  Along this W path 5 trowel size clods were dug at each of 5 locations; each clod was broken in half and put in a bucket, the other half of the clod was discarded.  The half-clods at each sample site were placed in a bucket and further broken in two.  All the quarter-clods from the eight sample sites were thoroughly mixed in two buckets and a subsample (ca 5kg) collected after mixing.  Soil samples were stored in sealed plastic bags at 5°C.

Samples from 47 paddocks were prepared for DNA analysis and a Pm baiting experiment.  After sieving (4 mm aperture), a 400g sub sample was sent to SARDI for DNA analysis. The remainder of each sample was slowly dried (72h @ 22-24C) then mixed with sand (40g soil + 112g sand), placed in a plastic cup (70mm width, 75mm height).  There were five replicates; soil from a Pm inoculated field trial (MET14) served as a control.  Four Sonali seeds were sown in each cup and the cups placed in a glasshouse (RCB design). The cups were watered to 22% Soil Moisture Content three times a week. After 18 days the cups were flooded for 48h then drained.  Seedlings were assessed for disease (chlorosis, stem cankers, death) three times a week.  Stem tissues were plated to isolate Pm.  Cultures with Phytophthora like growth on cornmeal agar were plated on low strength V8 agar and colony morphology, oospore production and oospore size used to identify Pm like cultures.  The isolation of Pm was attempted from all treatments that produced chlorosis followed by the appearance of Pm like stem cankers, in addition, the isolation of Pm was also attempted from any treatments where there were disease symptoms or seedlings with poor growth. The experiment was terminated after six weeks. 

Twenty six of the 47 soil samples produced symptomatic plants but Pm like cultures were isolated from only nine samples from growers paddocks and also from the MET14 control soil. Of the 48 paddocks soil (including the MET14 control soil), 11 had positive Pm DNA results.  Overall, most samples (9/11, 82%) which had positive DNA results yielded Pm cultures and most samples (36/37, 97%) which had negative DNA results did not yield Pm cultures (Table 2). 

Table 2. Comparison of Phytophthora medicaginis (Pm) DNA detection in 48 paddock soil samples and isolation success of Pm from Sonali chickpeas grown in these samples

Three samples gave contradictory results. One sample which yielded a Pm culture was negative for Pm DNA. Subsampling error may explain this result as this sample was part of a 5kg trowel collected sample; the 400g subsample used for Pm DNA analysis may not have contained Pm DNA. Two other samples were positive for Pm DNA but did not yield Pm cultures.  One of these had one seedling with a canker but Pm could not be isolated, in the other sample, seedlings in all 5 cups remained healthy.  These two samples had lower Pm DNA values (1,467 and 2,507 Pm copies/g soil) than all other samples (range 3022-872,069 Pm copies/g soil) except one (1,219 Pm copies/g soil).  Possible explanations for these results are: (i) more time may be required for symptoms to develop, or (ii) that the pathogen had died but some DNA had been detected.

The initial results are promising with an overall good correlation between PM DNA detection and PM isolation. However, further work is required to address some issues including subsampling effects.  

Pm DNA sampling in paddocks and disease risk determination

The DNA result for a soil sample from a paddock can only provide an indication of inoculum concentration and disease risk for the areas of the paddock which were sampled.  Therefore, the spread and locations of sampling across a paddock will affect how representative DNA results are for a paddock.  Because of the risk of rapid PRR disease build up following wet conditions it may be appropriate to treat a negative PreDicta B test result as indicating a low risk rather than a nil risk, as the pathogen could still be in areas of the paddock that were not sampled and so still cause PRR and reduce yield. 

To determine the risk of PRR disease it may be appropriate to target locations in a paddock where there is the best chance of Pm inoculum being present.  The pathogen thrives in soil with high moisture contents and so often occurs in low lying regions of paddocks where pooling following rain may occur.  The pathogen also carries over from season to season on infected chickpea volunteers, lucerne, native medics.  Including low lying areas and weedy areas of paddocks during PreDicta B^® soil sampling may provide the best strategy to predicting a paddocks disease risk of PRR in chickpea.

For detailed information on control of PRR in chickpea view the Chickpea Phytophthor Root Rot Management Northern Pulse Bulletin.

References

Ristaino, J. B., Larkin, R. P., & Campbell, C. L. (1993). Spatial and temporal dynamics of Phytophthora epidemics in commercial bell pepper fields. Phytopathology, 83(12), 1312-1320

Contact details

Sean Bithell & Kevin Moore
NSW Department Primary Industries
Ph: 0429 201 863 & 0488 251 866
Fx: 02 6763 1100
Email:  sean.bithell@dpi.nsw.gov.au & kevin.moore@dpi.nsw.gov.au

Acknowledgements

This research is made possible by the significant contributions of growers through both trial cooperation, paddocks access and the support of the GRDC, the authors would like to thank them for their continued support.  Thanks to Paul Nash, Gail Chiplin, Willy Martin and Kris King for technical support.

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