Evaluation of root-lesion nematode (Pratylenchus zeae) resistance assays for sugarcane accession lines

A total of 875 sugarcane accession lines were tested against P. zeae from 2011 to 2020 in nine trials at SRA Woodford Pathology Farm, Woodford, Queensland (26.9550° S, 152.7780° E) (Table 1). Among these, 722 lines (year 2011–2016) were from introgression populations and 153 lines (year 2017–2020) from Sugar Research Australia (SRA) core breeding germplasm populations. In total, 15 accession lines, including nine from commercial varieties/parents (including two susceptible controls), three introgression lines, and three Erianathus spp, were incorporated in multiple trials to obtain data on their resistance to P. zeae (Table 2).

Details of trial procedures and design of experiments were described elsewhere (Bhuiyan and Garlick, 2021; Bhuiyan et al., 2014; Croft et al., 2015). In short, all accession lines were collected from the SRA germplasm collection, situated at Meringa regional station, Queensland. Stalks were cleaned by stripping of leaves and sheaths, and cut into one-budded setts, and hot water treated at 50°C for 30 min to eliminate systemic diseases and placed in a germination chamber in trays with moist vermiculate. Germinated young plants were planted to pots, and transferred to a glasshouse for inoculation. Inoculation was done by applying approximately 2,000 nematodes per pot, and maintained for approximately 12 weeks before harvesting and assessing for resistance on number of nematodes per plant, and number of nematodes per g of roots. Root and shoot biomass were measured for each plant (Bhuiyan et al., 2016, 2019).

Nematodes were extracted from a mixture of root and potting mix using a modified Whitehead tray method (Whitehead and Hemming, 1965). Approximately 250 g of mixture was placed on double-layered Kleenex^® tissue paper (Kimberly-Clark Australia Pty., Mission Point, NSW, Australia) on a steel mesh set in a flat tray. The soil and roots were immersed in water and left for 48 hr at 25 ± 1°C. Nematodes were collected on a 38 μm sieve and then the extract was poured into a 30 ml plastic vial. Extracted nematodes samples were stored at 6°C until counting. Counting of nematodes were performed under a compound microscope (10X–40X) using a Peters counting chamber (Chalex Corporation, Wallowa, Oregon, USA) (Peters, 1952) of 1 ml capacity.

The number of nematodes per plant was recorded after the enumeration, and the number of nematodes per g of roots was estimated from the total number of nematodes present in total root biomass divided by the weight of the root system. Data were analysed by using linear mixed model to all dataset using Proc Mixed in SAS version 9.4 (SAS Institute, Cary, NC). For each trial accession lines were treated as fixed effects. Block (replication) and error term (residual) were treated as random effects. The interactions of the fixed effects were included in the model. Degrees of freedom were adjusted using the Kenward–Roger method (Kenward and Roger, 1997) and normality of residuals was tested using ProcUnivariate of SAS. Nematode counting data were ln(x + 1)-transformed before analysis. ProcCorr of SAS was used to calculate correlations from mean data between nematode resistance parameters and root or shoot biomass. Corr plot=matrix in SAS was used to calculate the correlation matrix to determine the linear relationship of root and shoot biomass with nematode damage parameters. On the basis of the linearity, ProcReg of SAS was used to determine the relationship between root biomass or shoot biomass and nematodes per g of roots. The percent of coefficient of variation (CV) within each trial for root and shoot biomass were calculated as CV = 100%(σ/µ), where σ and µ are the standard deviation and mean, respectively. As for the log-transformed data a formula, CV = 100 % e ln ( 10 ) 2 σ 2 ‒ 1 , where σ is the standard deviation of the log-transformed data, was used (Canchola et al., 2017). In total, 15 accession lines, as described earlier, were selected to determine the repeatability among the trials in relation to nematode resistance parameters using Pearson’s correlations (Table 2). Each accession line was included in at least four of the screening trials. SAS Corr in Fisher command was used to transform the data to normalize the distribution and stabilize the variance (Steel and Torrie, 1960).

The CVs for shoot biomass varied considerably from 17% in WFLN19-01 trial to 34% in Nep11-1 trial (Table 3). Root biomass had the highest variability among the all parameters in each trial, ranged from 31 to 57%. Least CVs were observed in nematode/plant (~3–6%), followed by nematodes/g root.

In 15 accession lines, correlation coefficients were based on common accessions tested between two trials. For both parameters, nematodes/plant and eggs/g root, 10 out of 28 possible combinations that include same accessions, correlations among trials were significant (p ≤ 0.05) (Table 4). In majority of the trial combinations correlations coefficients were not significant for root or shoot biomass. No differences in shoot biomass were observed among the 15 accessions except for Erianthus accession IJ76-370 and core accession Q232 (Table 2). Erianthus accession IJ76-370 produced significantly high root biomass compared to introgressions and core accessions. Relative nematodes/plant was significantly (p ≤ 0.05) low in three Erianthus lines compared to core accession lines, where three introgression lines had significantly low nematode numbers compared to two control accession lines Q208 and Q135.

Three out of seven trials showed moderate (r = 0.25–0.53, p ≤ 0.05) positive correlations between nematodes per plant and shoot biomass. Three out of nine trials had weak to moderate (r = 0.17–0.27, p ≤ 0.05) positive correlations and one had moderate negative correlations (r = ‒0.36, p ≤ 0.05) between nematode per plant and root biomass (Table 5). Nematodes per g of roots were negatively correlated (r = ‒0.23 to ‒0.31, p ≤ 0.05) with shoot biomass in four out of seven trials. Nematodes/g root were significantly (p < 0.001) negatively correlated with root biomass in all trials, and the value of correlations coefficients (r) ranged from ‒0.29 to ‒0.86.

The relationships between nematodes per g of roots with root biomass were consistently significant (p < 0.001) in all trials (Fig. 1). Except for one trial (Nep12-1), nematodes per g of roots explained 25–74% of variations in reduction of root biomass, and both intercepts and slops were highly significant (p < 0.0001) in all trials. In trial Nep12-1, nematodes per g of roots were able to explain only 9% of variation in root biomass, although, correlations and regression coefficients were significant (p < 0.001). In all trials 8–10% reductions of root biomass were estimated in each unit increase of log-nematode numbers per g of roots. The relationships between nematodes per g roots with shoot biomass varied among trials (Fig. 2). Four out of seven trials had significant (p ≤ 0.05) relationships of shoot biomass with nematode per g roots. Although, the regression coefficient of determination only explained 4–8% variations in shoot biomass. Relative nematodes/plant was significantly low in all Erianthus lines compared to core accessions, except for introgression lines. On the other hand, nematode/g roots were significantly low Erianthus lines compared to rest of the accession lines including introgression lines.

Figure 1:

Figure 2: