Vicia sativa

Dobosz, Renata, Flis, Łukasz, Bocianowski, Jan & Malewski, Tadeusz, 2023, Effect of Vicia sativa L. on Motility, Mortality and Expression Levels of hsp Genes in J 2 Stage of Meloidogyne hapla, Journal of Nematology 55 (1), pp. 1-17 : 3-4

Effect of V. Sativa seeds on motility and mortality of J 2 stage

SURfaCe STeRilizaTiOn Of V. SaTiva SeedS: The seeds were surface-sterilized by succesive immersion in 70% ethanol and in 7.3% sodium hypochlorite solution for 20 minutes each ( Sobczak et al., 2005). Then, the seeds were washed vigorously in distilled water for 30 minutes. Seeds prepared in this manner were used to make diffusates.

PRePaRaTiOn Of TOmaTO ROOT diffUSaTeS: Tomato ( SOlanUm lyCOPeRSiCUm L.) root diffusates were produced using the method elaborated by Devine et al. (1996). For this purpose, twenty tomato plants were grown in separate pots for four weeks. After this time, the pots were placed in funnels on funnel stands, which made it possible to collect the water which permeated the substrate, and to obtain the so-called soil filtrate. Then, the substrate with tomato roots was moistened by spraying its surface with 10 ml of distilled water. An additional 100 ml of distilled water was added to each pot. Excess water seeped through the substrate containing tomato roots and flowed out into a dish placed under the funnel. The first 50 ml of the filtrate collected from each pot, called tomato root diffusate, was mixed and used in the study of the motility and mortality of the J 2 stage.

PRePaRaTiOn Of COmmOn veTCH Seed diffUSaTeS: Eight types of diffusates were produced for the studies: two diffusates from surface-sterilized vetch seeds cultivars Ina and Jaga in water (Ina/Jaga surface-sterilized seeds + H 2 O), two diffusates from non-sterilized seeds of both vetch cultivars in water (Ina/Jaga seeds + H 2 O), two diffusates from surface-sterilized seeds of both vetch cultivars in the soil filtrate containing root secretions of tomato plants (Ina/Jaga surface-sterilized seeds + tomato root diffusates), and two diffusates from non-sterilized seeds of both vetch cultivars in the soil filtrate containing root secretions (Ina/Jaga seeds + root diffuses) .

In order to prepare the diffusate from surface-sterilized common vetch seeds, 1 gram of previously surface-sterilized V. SaTiva seeds (cv. Ina and cv. Jaga) was weighed and each of the weighed seed portions was immersed for 24 hours in 100 ml of distilled water. The diffusate from non-sterilized seeds of both common vetch cultivars (Ina and Jaga) was prepared in a similar manner, but with the use of non-sterilized seeds instead. Both the diffusate from surface-sterilized common vetch seeds, as well as the diffusate from common vetch seeds not subjected to surface sterilization in the soil filtrate, were produced in the same manner, except that instead of 100 ml of distilled water, 100 ml of soil filtrate was used.

STUdy Of THe effeCT Of diffUSaTeS On THe mOTiliTy Of THe J2 STage: Eight Petri dishes with a diameter of 3 cm were filled with 3 ml of each of the eight diffusates previously prepared, and 30 J 2 specimens of M. HaPla were transferred into each Petri dish. The dishes were placed at temperatures of 10, 17, and 21 ± 1° C. The control in the experiment were J2 stage specimens incubated in water and in soil filtrate. The experiment was performed twice, in six runs for each combination .

The nematodes were observed under a stereoscopic microscope 24 and 48 hours after exposure to seed diffusates. After this time, motile specimens and those that remained stationary after being touched with an entomological needle were counted ( Argentieri et al., 2008). The stationary specimens were placed in dishes with distilled water and observed again after 2 h and 24 h.

J 2 mORTaliTy aSSeSSmenT: For mortality assessment of J 2, stationary specimens were treated with 1 N NaOH according to the method by Chen and Dicson (2000). The percentage of stationary and dead specimens was determined using Abbott’s formula i=100×(1−nt/nc), in which i=% nematode immobility; nt=number of active nematodes after the treatment; and nc= number of active nematodes in the control (Argientieri et al., 2008).

STaTiSTiCal analySiS: The normality of the distribution of the four (immobile J 2 after 24 h, immobile J 2 after 48 h, immobile J 2 immersed in water and immobile J 2 after NaOH treatment) observed traits was tested using the Shapiro- Wilk normality test ( Shapiro and Wilk, 1965). The homogeneity of variance was tested using Bartlett’s test. Box’s M test tested multivariate normality and homogeneity of variance-covariance matrices. Non-normal traits were transformed using the power (Box-Cox) transformation with the lambda (λ) parameter at interval from −2 to 2 ( Kozak et al., 2008). Having the variables transformed and normally distributed, it was assumed that the data followed the multivariate normal distribution. A three-way (temperature, cultivar, treatment) multivariate analysis of variance ( MANOVA) was performed. Next, a three-way analysis of variance ( ANOVA) was carried out to determine the effects of temperature, cultivar, treatment and all interactions on the variability of all four observed traits. The mean values and standard deviations of traits were calculated. The Fisher’s least significant differences (LSDs) were calculated for individual traits, and on this basis, homogeneous groups were determined. The differences between combinations of the analyzed temperatures, cultivars and variants were verified by cluster analysis using the nearest neighbour method and Euclidean distances and presented as a dendrogram. The results were also analyzed using multivariate methods. A canonical variance analysis ( CVA) was applied to present a multi-trait assessment of the similarity of the tested combinations of temperatures, cultivars, and variants in a lower number of dimensions with the least possible loss of information. Mahalanobis distance was suggested as a measure of “polytrait” combinations’ similarity ( Seidler-Łożykowska and Bocianowski, 2012), the significance of which was verified by means of critical value Dα,called “the least significant distance” ( Mahalanobis, 1936). Mahalanobis distances were calculated for all combinations. The relationships between observed traits were assessed on the basis of Pearson’s correlation. Relationships of four observed traits were depicted in a heatmap. The GenStat v. 18 statistical software package ( VSN International) was used for the analyses.