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Revista mexicana de ciencias agrícolas

Print version ISSN 2007-0934

Rev. Mex. Cienc. Agríc vol.7 n.6 Texcoco Aug./Sep. 2016

 

Articles

Predictive capability of DAS-ELISA to barley stripe mosaic virus in wheat

Arturo Martínez-Mirafuentes1 

Noemí Valencia-Torres2 

Mónica Mezzalama2 

Mateo Vargas-Hernández2 

Ana María Hernández-Anguiano1  § 

1Colegio de Postgraduados-Fitopatología Carretera México-Texcoco km 36.5, Montecillo, Texcoco. C. P. 56230. Estado de México. Tel: 01 595 95 20200. Ext. 1606. (armami1276@hotmail.com; n.valencia@cgiar.org; m.mezzalama@cgiar.org; vargas_mateo@hotmail.com).

2Centro Internacional de Mejoramiento de Maíz y Trigo (CIMMYT), Carretera México-Veracruz, km 45, El Batán, Texcoco. C. P. 56237. Estado de México. Tel: 01 595 95 21900. Ext. 1242.

Abstract

Detection of pathogens associated with seed requires to count with representative and homogeneous samples to obtain reliable results. This research objective was to evaluate whether: 1) the level of infection (5, 10 and 15%); 2) the increase in the number of single sample (10, 15, 20 and 25) used to form composite samples; and 3) increasing and reducing the usual sample size from 10% (established at CIMMYT for diagnosis of pests and diseases) to 15 and 5%, affect the predictive capability of DAS-ELISA test to barley stripe mosaic virus (BSMV). From seed lots of bread wheat (Triticum aestivum L.), one naturally infected and another healthy, 210 single samples (MS), 20 g each, divided into equal groups of 70 MS samples with 5, 10 and 15% level of infection (NI) to form sample sizes (TM) of 15, 10 and 5%, and composite samples (MC) with 10, 15, 20 and 25 MS samples by NI and TM were formed. The results indicated that reducing the level of NI in MS samples from 15% to 10 and 5% decreased significantly (α= 0.05) in 21 and 31.5% respectively, the sensitivity of the test to BSMV virus. Similarly, reducing the number of MS samples to form MC samples significantly affected (α= 0.05) the sensitivity of the test to the virus and increased the likelihood of false negative record. Although the increase and reduction of TM from 10% to 15 and 5% significantly affected (α= 0.05) the sensitivity of the test to the virus, not finding a linear relationship between TM from the MC sample with the absorbance value. This study demonstrates the importance of the level of infection from the BSMV virus in the seed, of TM and MS number in MS in MC samples to obtain reliable results by DAS-ELISA.

Keywords: BSMV; sampling; seed

Introduction

The Barley stripe mosaic virus (BSMV), is a virus that mainly attacks barley, wheat and oat in most producing areas of North and South America, Asia, Africa, Europe and Australia (Mathre 1997; Bockus et al., 2010). Production losses and total yield of grain, caused by the disease, can be considerable, depending on the cultivar, level of infection, virulence of the virus and environment (Mckinney and Greeley, 1965; Carroll, 1986). Production losses are mainly due to decreased photosynthesis (Brakke et al., 1988) and induction of ovule and pollen sterility. Diseased plants with BSMV produce significantly less spikes and seeds than healthy, with decrease in weight and total grain yield (up to 35%).

BSMV is spread by seed, pollen, ovule and sap but not by vector insects such as aphids (Carroll, 1972; Carroll, 1974; Carroll and Mayhew, 1976). In barley transmission is by seed via ovule but in wheat is by pollen and natural infections, in this crop, resulting from the transfer of sap by contact between plants. In different hosts seed transmission is relatively inefficient.

Because the virus can remain viable for several years in the seed this is constituted as the source of primary inoculum and the main route of transmission (Carroll, 1986). It is mentioned that in early infections some virus races may have 100% transmission by seed, but the most common is 50 to 60% transmission. Seed transmission can be determined directly by the search of symptoms development in seedlings grown in greenhouses because in general infected seeds with BSMV are asymptomatic (Carroll, 1980; Mathre, 1997; Bockus et al., 2010).

For the detection and identification of BSMV different methods are used. Field, plant inspection to check for characteristic symptoms of the disease; in suspect plants, observation of leave preparations in electron microscope for the presence of viral particles characteristics; and in leaf and seed, by serological tests such as enzyme-linked immunosorbent assay (ELISA) with specific antibodies, or with the polymerase chain reaction (PCR) test with specific primers (Jackson et al., 1991).

Detection and timely identification of BSMV are essential for disease control of barley stripe mosaic and important for its regulation and control through phytosanitary certification and quarantine programs in domestic and international seed trade (Morrison, 1999). As indicated above objectives were established to determine whether: 1) the level of infection (from 5, 10 and 15%); 2) the increase in the number of single sample (10, 15, 20 and 25) used to form composite samples; and 3) increasing or reducing the size of the usual sample 10% to 15 to 5%, affects the sensitivity of DAS-ELISA for detection of BSMV. The hypotheses were: a) levels of infection from 5, 10 and 15%; b) the increase in the number of single samples used to form composite samples; and c) increase or reduction of current sample size of wheat from 10% to 15 and 5% do not affect the sensitivity of DAS-ELISA.

Materials and methods

DAS ELISA test establishment

DAS (double-antibody sandwich) - ELISA test is a variant of direct ELISA test, developed for the detection of plant viruses with a specific enzyme-antibody conjugate for each particular virus. In this case the test was established with samples of ground seed (2 g of seed) in 96-well polystyrene plates. The reagents used thus the protocol were from Agdia®, DAS Elisa (2013) for BSMV.

Sensitization and plate washing

The antibody coverage was diluted 1: 200 for this where mixed 10 000 µl of the coverage buffer (1X), pH 9.6, with 50 µl of antibody coverage concentrated; except for blank wells, in each well 100 µl of antibody dilution coverage were added and incubated for 4 h at room temperature or overnight at 4 °C, in humid chamber. After incubation, the content of the plate was emptied and washed with PBST (1X) buffer wash with a multichannel wash bottle. This process was repeated three times and between each wash the plate was strongly shacked down on a folded paper towel to remove excess liquid and bubbles.

Loading samples

To each of the tubes with ground seed samples were added 20 ml of extraction buffer (1X) 1:10 ratio (sample weight in g: buffer volume in ml). Except blank wells, added 100 µl per well of each sample per duplicate, thus positive control, negative control and the buffer according to the preset load diagram. The plate was incubated for 2 h at room temperature or overnight at 4 °C, in humid chamber. Upon completion of the incubation the plate was emptied and washed eight times with wash buffer, in order to remove any residue from the sample. Between each wash the plate was shacked, down on a folded paper towel to remove excess liquid and bubbles.

Addition of enzyme conjugate

Shortly before using the enzyme conjugate (alkaline phosphatase) it was diluted to a ratio 1: 200. For this 10 000 µl of ECI buffer (1X) with 50 µl of enzyme conjugate concentrate were mixed; except blank wells, in each well 100 µl of enzyme dilution were added. After 2 h incubation in a moist chamber, the plate was emptied and washed eight times with wash buffer, in order to remove any residual sample. Between each wash the plate shacked, down on a folded paper towel to remove excess liquid and bubbles.

Reveil plate

For revealed a PNP (p-nitrophenyl phosphate) solution at 1 mg per ml with two PNP tablets (substrate) dissolved in 10 ml PNP buffer (1X) in a shaker Vortex Scientific Industries G-560 was prepared. It avoided touching the tablets and expose the PNP solution to intense light to avoid contamination and false positive development by color development in the wells of negative samples. Immediately after preparation, 100 µl of the PNP solution were added in each plate well and incubated in the dark at room temperature for 60 min. Elapsed time of incubation, 70 µl of sodium hydroxide (NaOH) 3 molar per well were placed to stop the reaction in the plate. Reading the plate was performed in a BioTek ELx808 spectrophotometer at 405 nm.

Preparation of single samples

From two seed batches of bread wheat (Triticum aestivum L.), one naturally infected seed (SI) with barley stripe mosaic virus (BSMV and healthy seed (SS), single samples (MS) 20 g of seed were formed. Each MS sample was prepared according to the level of infection (NI): NI 15%, 3 g of SI with 17 g of SS; NI 10%, 2 g of SI with 18 g of SS; and NI 5%, 1 g of SI with 19 g of SS. Once MS samples are formed these were deposited separately in paper envelopes properly identified. In total 210 MS samples (70 MS samples per NI) were prepared of which 2 g of seed each were taken to be ground and analyzed separately by DAS-ELISA according to the protocol described above.

Preparation of sample size and composite samples

Of each of the 210 MS samples were taken separately, 1.0, 1.9 and 2.56, g of seed to form sample sizes (TM) of 5, 10 and 15% respectively (Figure 1). For this from an initial batch of 20 g, 1 g was taken to form TM of 5%; the remaining 19.0 g, 1.90 g were taken to form TM 10%; and from the remaining 17.1 g, 2.56 g were weighted to form TM 15%.

Figure 1. Diagram for composition of single samples (MS) with their level of infection and structuring of composite samples (MC= NMS + TM + NI). 

The composite samples (MC) were formed with different numbers of samples MS (25, 20, 15 and 10 NMS). In total 36 MC samples, each MC sample with three NI and three TM and a total weight according to TM (Figure 1) were formed. For practical management purposes, the above MC were labeled considering first the code to number of MS samples, then the code for TM and last NI. For example, the sample labeled MC25-15-15 indicates that is about a MC sample with 25 MS samples with TM 15% and NI 15%. While the MC25-10-15 indicates that is a MC sample of 25 MS samples with TM 10% and NI 15%. While MC25-5-15 indicates that is a MC sample with 25 MS samples, with TM 5-% and NI 15%. This methodology was applied to the remaining 33 samples.

Figure 1 and Table 1 illustrates the formation scheme MS and MC samples.

Table 1. Formation of composite samples (MC) with different levels of infection (NI) and sample size (TM). 

Muestra MC Núm. muestras MS NI 5, 10 y 15% TM 5, 10 y 15% Total muestras MC*
MC25 25 3 3 9
MC20 20 3 3 9
MC15 15 3 3 9
MC10 10 3 3 9

Evaluation of the number of MS, TM and NI on composite samples

To determine whether NMS, the increase or reduction in the size TM and NI level that make MC samples affects the sensitivity of DAS-ELISA test to BSMV, these were analyzed separately each of the 36 MC samples. Of each MC sample, three replications of 2 g of seed each (108 subsamples in total) were taken, which were deposited in falcon tubes type of 50 ml properly identified to be ground separately in a mill Peter Instruments. Ground seed was recovered in the same tube. Between each grinding the mill was cleaned with compressed air and seed fragments stuck in the mill discs were removed with a dissecting needle to prevent contamination between samples. The procedure for analysis of the subsamples was made as described in the establishment of DAS-ELISA test for sensitization and plate washing, adding samples, addition of the enzyme conjugate and plate reveil, Agdia protocol, Das Elisa (2103) for BSMV.

Statistical analysis

Absorbance data obtained from the evaluation of MS and MC samples were analyzed with the statistical program SAS system for Windows 9.4, using the analysis of variance the lineal model corresponding to a randomized complete block design (RCBD) with sub - sampling, with the procedure of general linear model (GLM) and multiple comparisons test using mean honest significant difference (HSD) from Tukey, with a significance level of 5% (α= 0.05).

Results and discussion

NMS and NI effect on sensitivity of DAS ELISA test

Table 2 shows the average values and mean comparison of absorbance in different NMS used to form MC samples with different NI from BSMV, using honest significant difference (HSD) of Tukey with a significance level of 5% (α= 0.05). With levels 15, 10 and 5% NI recording samples with values above the threshold value (0.22). Similarly, the mean values per group and general means were also higher than the threshold. However, when analyzing the results by level and between levels differences were found.

Table 2. Mean values and comparison of mean absorbance in different NMS used to form MC samples with different NI from BSMV. 

NI (%) NMS NI (%)
NMSx 15 10 5 25 0.74 by 15 0.92 ay
25 0.97 0.7 0.54 20 0.72 b 10 0.73 b
20 0.89 0.57 0.71 15 0.85 a 5 0.63 b
15 0.91 1.05 0.6 10 0.74 b
10 0.87 0.63 0.72 DSH 0.032 DSH 0.107

The overall average value (0.92) recorded with NI level 15% was significantly different (α= 0.05) by to that registered with NI 10 and 5%, which showed statistically similar values (0.73 and 0.63, respectively). This indicates that by reducing the NI level from 15% to 10 and 5% was detected a percentage of decrease of 20 and 34%, respectively, of the sensitivity of DAS-ELISA test to BSMV. These results confirm the results found by Scott and Zummo (1995) who report that as the level of natural incidence of a pathogen is reduced in the seed the likelihood of not detecting it increases.

By analyzing the comparison of means of absorbance at different NMS, it was found that in NMS of 25, 20 and 10 forming the MC samples have no statistically significant differences (α= 0.05) except for NMS 15 (0.85) that showed differences with the previous. When comparing the NMS with lower absorbance value to the threshold (0.22), samples with NI 10 and 5% recorded the highest number of negative MS samples (14 and 24 samples, respectively) compared to samples with NI 15% (6 samples). That is, as NI decreased from 15% to 10 and 5% the number of negative samples to BSMV increases (data not shown). In short with a level of infection of 15% a percentage of 91.5% probability of detecting BSMV by DAS-ELISA test was detected; that is, from the 70 MS samples with NI 15%, 64 of them were positive to the virus; instead with NI 10% detected a percentage of 80% and with NI 5% detected a percentage of 66% probability of detecting BSMV.

The level of infection of a seed or incidence of the pathogen in the crop is key for its detection (Scott and Zummo, 1995) also determines the sample size or composite sample, as found by Priou et al. (2001), that reducing the composite sample size of potato tubers are based on the level of incidence of Ralstonia solanacearum in crops.

Effect of NMS, TM and NI on composite sample

Table 3 shows the absorbance average results of 36 MC with DAS-ELISA test. Overall average absorbance values of MC samples were above the threshold value of absorbance (0.21) only in the sample MC10-10-5 the absorbance value (0.18) remained below the threshold, as in 4 of 6 individual observations (replications) obtained a lower absorbance than threshold. Although MC10-15-15, MC10-10-10 and MC10-5-5 recorded two replications below the threshold, by averaging the six replications this exceeded the absorbance threshold, marking it as positive to BSMV. This indicates that when MC samples are formed with a NMS lower to 15 or equal to 10 decreases the sensitivity of DAS-ELISA test to BSMV and the probability of recording false negatives increases.

Table 3. Average absorbance values obtained with DAS-ELISA test of MC samples of wheat seed. 

TM NI NMS (Valor promedio de absorbancia)
25 20 15 10
15 15 3.23 2.95 1.84 0.71
10 15 2.81 3.36 2.12 0.93
5 15 3.08 2.33 2.25 0.52
15 10 1.38 3.25 2.61 0.66
10 10 1.54 0.58 1.7 0.75
5 10 2.19 2.35 2.13 1.43
15 5 2.39 1.48 1.07 1.32
10 5 2.72 0.91 0.95 0.18
5 5 2.8 1.75 0.84 0.58

Because when comparing the 36 values showed in Table 3, obtained from the combination of the three factors, as if they were individual treatments does not make much sense, an analysis of variance was performed considering the factorial structure of the treatments, 3 factors: i) number of single samples (NMS) with 4 levels (25, 20, 15 and 10); ii) Sample size (TM) with three levels (15, 10 and 5); and iii) level of infection (NI) with three levels (15, 10 and 5). Table 4 shows the results of multiple comparisons of means using honest significant difference (HSD) Tukey significance level of 5% (α= 0.05), for each of the principal effects. Now the averages presented come from a higher number of individual values (N).

Table 4. Multiple comparisons of mean of absorbance for the principal effects of the factors number of single samples (NMS), sample size (TM), and level of infection (NI) used for the formation of composite samples (MC). 

NMS TM NI
MC Media (%) Media (%) Media
25 2.46 ax 15 1.91 a 15 2.18 a
20 2.11 a 10 1.55 b 10 1.71 b
15 1.72 b 5 1.85 a 5 1.42 c
10 0.79 c
DSH 0.365 DSH 0.288 DSH 0.288
N 54 N 72 N 72

The results from analysis of the comparison of means for the overall principal effect of absorbance between MC samples with different NMS, TM and NI indicate statistical differences (α= 0.05) between samples. Specifically highlights differences between the averages recorded by MC samples with NMS 25 (2.46a) and NMS 20 (2.11a) with MC samples with NMS 15 (1.72b) and NMS 10 (0.79c) and differences between the latter. This indicates that as NMS decreases in the MC sample, statistically decreases the absorbance value, confirming the findings by Fernández (2007) mentioning that large sample sizes recorded higher confidence level and degree of accuracy in contrast to what was presented by small samples.

When comparing the mean absorbance between MC samples with different TM statistical differences (α = 0.05) between samples were found. It is noteworthy that these differences occurred only between MC samples with TM 15 (1.9a) and 5 (1.8a) with MC with TM 10 (1.5b). That is, between the sizes of 15 and 5% are no statistical differences but between these and the size 10% are significant differences. This may be related to the fact that MC10-10-5 sample had two samples (replications) with value (0.15 and 0.13) below the threshold (0.21).

In this case a linear relationship between TM from MC sample with the absorbance value was not found; that is, there is no tendency between means from composite samples according to sample size. These results agree with those found by Moreno and Castillo (1978) for classification of coffee trees, who indicate that a sample of 250 g used for grain size can be reduced to 100 or 50 g without causing any alteration. As well as those reported by Thomas et al. (2005) who mentioned that there is no difference in the infection rate between 40 and 80 composite samples obtained from wheat batches of 200 and 500 t, respectively, infected with Microdochium nivale.

Several researchers point out that the analysis of small sample sizes results in lower confidence level and degree of accuracy in contrast with those registered with larger sample sizes which record better results (Lawrence et al., 1995; Fernández, 2007; Miyamoto et al., 2008; Montesinos et al., 2010). However, Thomas et al. (2005)) indicate that infected seed batches with Tilletia tritici is the level of infection and not the NMS from MC samples which determines the capability of a test to detect the fungus.

According Nyrop et al. (1999) when sampling plans are developed it should start by checking what is known about the distribution of sampling and perform a sensitivity analysis to determine whether a refinement of this information is justified, and that the sample size should be based on information from the representative sample, as this is a good indicator of the incidence of pest management unit.

By analyzing the means for the principal effects of absorbance between MC samples with different NI statistical differences (α= 0.05) between the samples were found. In this case means of 2.18a, 1.71b and 1.42c for MC samples with NI 15, 10 and 5%, respectively (Table 4) were recorded. As NI decreased, the absorbance value from MC sample significantly decreases. Figure 2 shows the linear trend of the average absorbance values of MC samples according to NMS, TM and NI with only the MC10-10-5 value below the threshold line.

Figure 2. Trend of average absorbance values of composite samples (MC) of wheat seed. Each MC sample was formed with different number of single samples (NMS, 25, 20, 15 and 10), sample size (TM, 15, 10 and 5%) and level of infection (NI, 15, 10 and 5%) from BSMV. The values from the brown line indicate the average of three MC samples; dotted purple, the linear trend of values; and blue, the average absorbance threshold (0.21). 

Priou et al. (2001) found that as the level of disease incidence by Ralstonia solanacearum decreases in culture, fungus detection decreases in tuber. Therefore these authors suggest to analyze large composite samples when disease levels are low in the crop. Similarly, Scott and Zummo (1995) observed that when the level of natural infection with Aspergillus flavus is high (3%) the size of sample to be analyzed is reduced but when the level of infection is very low not even with larger sample sizes detects the fungus.

The results described above are evidence of the importance that should be given to sampling as a fundamental activity in a seed testing program (Morrison, 1999). ISTA (2013) points out that a batch can be composed of harvested seeds from a single field or several sections of land and the objective of sampling from seed will be to obtain a representative sample of adequate size so that the results of the laboratory analysis reflect the quality of the seed batch. Knowing the importance that parameters of sample size and level of infection in samplings of seed batches is essential to determine the probability of selecting positive or negative samples to the pathogen of interest which will result in reliability of results and in making decisions about the final destination of the batch assessed.

Conclusions

The level of infection (NI 15, 10 and 5%) and increased number of single sample (NMS 10, 15, 20 and 25) used to form composite samples; and increasing and reducing the usual sample size (TM) from 10% to 15 and 5% respectively, affected the sensitivity of DAS-ELISA test for detection of barley stripe mosaic virus (BSMV) in wheat seed.

By reducing NI in single samples (MS) from 15% to 10 and 5% it decreased significantly (α= 0.05) in 21 and 31.5%, respectively, the sensitivity of the test to virus. Similarly, reducing NMS to form composite samples (MC) significantly (α= 0.05) affected the sensitivity of the test to the virus and increase the likelihood of recording false negative. The increase and reduction of TM from 10% to 15 and 5% significantly (α= 0.05) affected the sensitivity of the test to the virus but no linear relationship was found between TM of MC sample with the absorbance value. Among the sizes 15 (1.91a) and 5% (1.85a) no statistical differences (α= 0.05) were found but between them and size of 10% (1.55b) there are, indicating that there is a tendency between means of MC samples according to sample size.

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Received: March 2016; Accepted: June 2016

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