Plant-parasitic nematodes are major constraints to sugarcane production worldwide (Ramouthar and Bhuiyan, 2018). In Australia, plant-parasitic nematodes cause 5 to 20% yield loss per year, costing over $80 million in productivity (Blair and Stirling, 2007). The most important nematodes of sugarcane in Australia are root-lesion nematode (
Rotation crops or fallowing provide short-term protection, but
Modern sugarcane varieties were derived from crosses between noble cane
A collaborative research project between Australia and China in the late 1900s and early 2000s used new sources of wild relatives of sugarcane,
The parameter(s) used to screen progeny for disease resistance needs to be realistic, and repeatable. The reproductive capabilities of nematodes on sugarcane accession lines were used to measure the resistance to nematodes in screening trials. In this process, each test line was inoculated with 5,000 eggs of
Scoring the visual symptoms of nematode infection (e.g., galling) has been used by some researchers to rapidly rate plants for resistance to nematodes, particularly when the symptoms are obvious. Wang and Goldman (1996) used a 0 to 5 scale to rate the severity of galling on carrots inoculated with
This study examined some of the parameters used to evaluate root-knot resistance in sugarcane accession lines from 2012 to 2019 in Australia to determine suitable parameters that provide reliable and repeatable results.
A total of 802 sugarcane accession lines were tested against
Trial names, trial year, number of accession lines and types of sugarcane populations screened for root-knot nematode (
Trial name | Trial year | Number of accession lines | Source |
---|---|---|---|
Nem19-1 | 2019 | 42 | Core breeding program |
Nem18-1 | 2018 | 56 | Core breeding program |
Nem17-1 | 2017 | 56 | Core breeding program |
Nem16-2 | 2016 | 95 | Introgression population |
Nem15-1 | 2015 | 112 | Introgression population |
Nem13-1 | 2013 | 138 | Introgression population |
Nem13-2 | 2013 | 155 | Introgression population |
Nem12-1 | 2012 | 148 | Introgression population |
Total accession lines | – | 802 | – |
List of 22 sugarcane accession lines used in multiple trials with root biomass, and nematode resistance parameters.
Accession | Type | No. of trialsa | Root biomass (g) | Relative eggs per plante (%) | Relative eggs per g root (%) | Visual gall rating |
---|---|---|---|---|---|---|
IJ76-333 |
|
5 | 25.3±12.5d | 12.4±2.19 | 25.6±18.99 | 1.3±0.06 |
IJ76-370 |
|
5 | 23.2±11.16 | 10.7±1.19 | 7.8±3.26 | 1.1±0.1 |
KQ228 | Core | 8 | 21.0±7.66 | 43.2±6.62 | 23.6±9.1 | 2.6±0.17 |
Q135c | Core | 7 | 12.9±4.78 | 125.2±27.71 | 145.0±67.32 | 3.2±0.2 |
Q138 | Core | 7 | 15.2±4.61 | 40.0±8.68 | 25.5±10.19 | 2.7±0.35 |
Q183 | Core | 4 | 6.8±4.14 | 67.8±11.13 | 74.9±37.44 | 3.0±0.19 |
Q200 | Core | 6 | 10.9±5.07 | 144.2±43.27 | 115.7±40.94 | 2.9±0.2 |
Q208c | Core | 8 | 16.2±5.02 | 77.9±8.56 | 60.6±28.92 | 3.0±0.23 |
Q231 | Core | 4 | 17.6±12.07 | 36.4±14.26 | 16.6±10.16 | 3.0±0.51 |
Q232 | Core | 6 | 14.3±5.13 | 55.5±14.06 | 43.8±21.41 | 2.7±0.31 |
Q238 | Core | 4 | 16.2±7.96 | 53.5±14.84 | 25.8±12.79 | 3.4±0.18 |
Q240 | Core | 5 | 20.4±6.38 | 101.4±25.49 | 29.0±13.63 | 2.8±0.3 |
Q241 | Core | 4 | 8.0±6.59 | 30.8±8.73 | 88.1±49.45 | 3.1±0.43 |
Q242 | Core | 4 | 11.8±6.31 | 64.6±28.2 | 30.5±14.41 | 3.0±0.57 |
Q245 | Core | 6 | 9.4±4.78 | 45.7±7.38 | 58.9±28.38 | 2.7±0.25 |
Q248 | Core | 4 | 16.2±8.99 | 139.2±35.96 | 51.5±24.73 | 3.3±0.28 |
Q249 | Core | 4 | 13.1±7.65 | 24.6±3.16 | 17.0±10.41 | 3.0±0.34 |
Q251 | Core | 4 | 12.6±4.82 | 90.1±21.37 | 30.7±11.44 | 2.7±0.37 |
QBYN05-20563 | Introgression | 4 | 24.1±3.03 | 11.7±3.4 | 3.7±1.83 | 2.6±0.22 |
SRA1 | Core | 4 | 6.2±3.65 | 43.2±11.22 | 81.7±57.66 | 2.7±0.24 |
SRA2 | Core | 4 | 3.5±1.66 | 43.9±12.05 | 79.9±46.09 | 2.4±0.14 |
SRA3 | Core | 4 | 8.5±5.13 | 60.7±14.42 | 89.0±63.14 | 2.9±0.4 |
Details of trial procedures and design of experiments were described elsewhere (Bhuiyan et al., 2014, 2016; Croft et al., 2015). In short, all accession lines were collected from the SRA germplasm collection, Meringa, Queensland. Stalks of accession lines were stripped of leaves and leaf sheaths, cut into one-budded setts using an electric saw, hot water treated at 50oC for 30 min to eliminate systemic fungal and bacterial diseases and placed in a germination chamber in trays with moist vermiculite. Germinated young plants were planted to 700 ml pots filled with sterilized fine white washed sands (Caboolture Gravel & Landscapes, Morayfield, Qld), maintained in a glasshouse for inoculation. Each trial was set out on air conditioned benches using a randomized complete block design in five replicate pots, and one plant per replication. Two data loggers (Thermodata Pty Ltd, Brisbane, Australia) were included in each trial by burying them approximately 5 cm deep in potting media to record the temperature of the potting media during the trial period. Two commercial varieties Q208 and Q135 that were highly susceptible to
For the extraction of eggs, roots were washed free of sands, submerged in 1% NaOCl (bleach) and agitated for 5 min. The bleach solution was then poured through two sieves (a 150 μm sieve over a 38 μm sieve), with eggs being collected on the lower sieve, and finally washed into a 30-ml plastic vial. Extracted egg samples were stored at 6°C until counting. Enumeration of a subsample of nematode eggs were performed under a compound microscope (10×- 40×) using a Hawksley slide counting chamber of 1 ml capacity.
The number of eggs per plant was recorded after the enumeration, and the number of eggs per g of root was estimated from the total number of eggs present in the pot divided by the fresh weight of the root system. Data were analyzed by fitting a linear mixed model to all datasets using
The CVs for sugarcane biomass and nematode parameters varied among trials. The highest variations were in root biomass, ranging from 30 to 49% (Table 3). The CVs in the nematode parameters, eggs/plant, and eggs/g roots were higher in the early trials in years 2012 (Nem12-1) and 2013 (Nem13-1 and Nem13-2), ranging from 25 to 47%. In all other trials, the nematode parameter, eggs/plant had the lowest variations ranged from 2.3 to 9.2%, followed by eggs/g root (3.2-12%).
Coefficient of variance (%) among shoot biomass, root biomass, visual ratings, number of eggs per plant and number of eggs per g of roots in each trial.
Trial name | Shoot biomass | Root biomass | Visual rating | Eggs per plant | Eggs per g root |
---|---|---|---|---|---|
Nem19-1 | 21.5 | 36.3 | 20.5 | 2.3 | 6.4 |
Nem18-1 | 20.7 | 38.7 | 19.3 | 2.8 | 3.2 |
Nem17-1 | 27.3 | 48.7 | 20.5 | 2.7 | 9.9 |
Nem16-2 | - | 38.2 | 22.1 | 6.0 | 8.7 |
Nem15-1 | 20.9 | 38.3 | 30.0 | 9.2 | 11.9 |
Nem13-1 | 21.5 | 29.6 | 22.4 | 28.6 | 47.1 |
Nem13-2 | 27.9 | 37.0 | 22.6 | 25.3 | 27.8 |
Nem12-1 | 24.0 | 37.5 | 29.3 | 25.0 | 25.5 |
In 22 accessions, eggs per plant were more repeatable than eggs per g of root and visual ratings across the trials (Table 4). In 18 out of 28 possible trial combinations that include the same accessions, correlation of eggs per plant among trials were significant (
Pearson correlation coefficients among 22 accession lines to measure the repeatability among trials in relation to nematode (
Trial | by Trial | No. of accession linesa | Eggs per plantb | Eggs per g root | Gall rating |
---|---|---|---|---|---|
Nem12-1 | Nem13-1 | 6 | 0.99*** | 0.89* | 0.92** |
Nem12-1 | Nem13-2 | 6 | 0.98*** | 0.94** | 0.86* |
Nem12-1 | Nem15-1 | 6 | 0.10 ns | 0.20 ns | 0.56 ns |
Nem12-1 | Nem16-2 | 7 | 0.98*** | 0.76* | 0.20 ns |
Nem12-1 | Nem17-1 | 6 | 0.94** | 0.95** | 0.86* |
Nem12-1 | Nem18-1 | 8 | 0.93** | 0.63 ns | 0.64 ns |
Nem12-1 | Nem19-1 | 8 | 0.87** | 0.92** | 0.86** |
Nem13-1 | Nem13-2 | 3 | 0.99 ns | 0.82 ns | – |
Nem13-1 | Nem15-1 | 12 | 0.47 ns | 0.31 ns | 0.27 ns |
Nem13-1 | Nem16-2 | 12 | 0.92*** | 0.10 ns | 0.35 ns |
Nem13-1 | Nem17-1 | 10 | 0.74* | 0.60 ns | 0.72* |
Nem13-1 | Nem18-1 | 8 | 0.91** | 0.71* | 0.82* |
Nem13-1 | Nem19-1 | 7 | 0.69 ns | 0.59 ns | 0.89** |
Nem13-2 | Nem15-1 | 5 | −0.02 ns | 0.33 ns | 0.71 ns |
Nem13-2 | Nem16-2 | 6 | 0.95** | 0.80 ns | −0.30 ns |
Nem13-2 | Nem17-1 | 5 | 0.92* | 0.95* | 0.68 ns |
Nem13-2 | Nem18-1 | 6 | 0.90* | 0.46 ns | 0.68 ns |
Nem13-2 | Nem19-1 | 6 | 0.86* | 0.83* | 0.87* |
Nem15-1 | Nem16-2 | 18 | 0.19 ns | 0.29 ns | −0.30 ns |
Nem15-1 | Nem17-1 | 15 | 0.25 ns | 0.12 ns | 0.19 ns |
Nem15-1 | Nem18-1 | 10 | 0.51 ns | 0.66* | 0.16 ns |
Nem15-1 | Nem19-1 | 13 | 0.66* | 0.60* | -0.48 ns |
Nem16-2 | Nem17-1 | 14 | 0.74** | 0.28 ns | 0.06 ns |
Nem16-2 | Nem18-1 | 10 | 0.81 ns | 0.63* | −0.38 ns |
Nem16-2 | Nem19-1 | 12 | 0.43 ns | 0.40 ns | 0.27 ns |
Nem17-1 | Nem18-1 | 8 | 0.97*** | 0.77* | 0.82** |
Nem17-1 | Nem19-1 | 13 | 0.92*** | 0.86*** | 0.79** |
Nem18-1 | Nem19-1 | 10 | 0.93*** | 0.71* | 0.88** |
There were no significant correlations of nematode eggs per plant with root and shoot biomass in any of the eight trials (Table 5). Nematode eggs per g of root were negatively correlated (
Pearson correlation coefficients to compare relationships of nematode (
Eggs per plant | Eggs per g root | Visual rating | ||||
---|---|---|---|---|---|---|
Trial name | Shoot biomass | Root biomass | Shoot biomass | Root biomass | Shoot biomass | Root biomass |
Nem19-1 | −0.08 nsa | −0.27 ns | −0.47** | −0.74*** | −0.14 ns | −0.41** |
Nem18-1 | 0.01 ns | −0.03 ns | −0.36** | −0.45** | 0.01 ns | −0.08 ns |
Nem17-1 | 0.070 ns | −0.26 ns | −0.42** | −0.65*** | −0.004 ns | −0.26 ns |
Nem16-2 | – | 0.13 ns | – | −0.44*** | – | 0.11 ns |
Nem15-1 | −0.093 ns | −0.07 ns | −0.24** | −0.28** | −0.19 ns | −0.12 ns |
Nem13-1 | 0.15 ns | 0.14 ns | 0.12 ns | −0.06 ns | 0.30** | 0.21* |
Nem13-2 | 0.19 ns | 0.02 ns | 0.08 ns | −0.19* | −0.10 ns | −0.40*** |
Nem12-1 | 0.18 ns | 0.06 ns | −0.02 ns | −0.23** | 0.24* | −0.04 ns |
The relationships between eggs per g of root and root biomass varied among the trials (Fig. 2). In the trials Nem19-1 and Nem17-1 eggs per g of root explained 58% of variations in root biomass, and the regression and correlation coefficients were highly significant (
A reliable parameter for assessing a screening trial depends on two important criteria, low variation within a trial and repeatable among trials. This study clearly indicated that nematode eggs per plant showed higher repeatability across the trials, and relatively low variations (CV) compared to other parameters. Between two other nematode resistance parameters, nematode eggs per g roots showed greater reliability and repeatability compared to visual gall rating.
Root-knot nematode eggs per g of root on a wide range of sugarcane introgression and commercial breeding lines were negatively correlated with root biomass in all but one trial and with shoot biomass in four out of the eight trials. In contrast, eggs per plant was not significantly correlated with shoot or root biomass in any trial. Visual rating of galling on plants was significantly correlated with shoot or root biomass in less than half of the trials and the correlation was not consistently negative. Sugarcane breeders evaluate large numbers of accessions each year for nematode resistance. It is important to decide which assessment parameter is appropriate and provides reliable results without delay. Resistance selection should be based on the ability of an accession line to suppress reproduction and maintain shoot and root biomass. The regression analysis indicated that nematode eggs per g of root were an indicator of varietal resistance for sugarcane to root-knot nematodes (Fig. 2).
Nematode eggs per plant has been used widely to measure the reproductive capabilities of nematodes on test plants in resistance screening trials (Bhuiyan et al., 2019; Stirling et al., 2011; Thompson et al., 2009). In our trials this parameter provided better repeatability in separating accession lines with relatively low variations in the breeding program (Table 4). As indicators for resistance, egg numbers per plant was more effective and independent of root biomass of the accessions. The negative correlations of nematode per g of root with root biomass can be useful in separating accession lines on the basis of this parameter. However, the greater variations of root biomass within an accession line, manifested in this study, rendered it less repeatable. Variation in root biomass is common among sugarcane varieties in Australia (Pierre et al., 2019).
Both the parameters eggs per plant and eggs per g root were able to separate accession lines accurately (Table 2). Two wild sugarcane accessions IJ76-333 and IJ76-370 and introgression line QBYN05-20563 showed a high level of resistance reaction to
Visual ratings for root-knot nematode galling were highly correlated with eggs per plant and eggs per g of root. This is in agreement with our previous work where visual ratings were correlated with extracted eggs from the sugarcane accession lines (Bhuiyan et al., 2014). Although this parameter was less reliable and repeatable compared to two other resistance parameters, visual rating has been used to screen a range of crops such as peanuts and
Shoot and root biomass can be used to assist in comparisons between accessions (Stirling et al., 2011). As an obligate parasite of plants, reproduction of
Greater populations of
There were moderate to strong correlations for eggs per plant in accession lines among majority of the trials (Table 4), suggesting that this parameter is the most reliable indicator to assess root-knot nematode resistance in sugarcane. However, the number of eggs per g of sugarcane root was clearly related to root and shoot biomass, which may be indicative of both resistance and tolerance. Therefore, eggs per g of root may be used as a secondary selection factor for advancing germplasm in a breeding program. Further research is required to verify the resistance of sugarcane accession lines in the glasshouse and under field conditions.