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 : 5-10

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

The results of the MANOVA performed indicated that all factors [temperature ( T): Wilk’s λ=0.2932; F =83.19; cultivar ( C): Wilk’s λ=0.3015; F =227.60; variant ( V): Wilk’s λ=0.0705; F =79.86] and all interactions [ T × C: Wilk’s λ=0.7850; F =12.64; T × V: Wilk’s λ=0.0894; F =33.22; C × V: Wilk’sλ=0.2618; F =32.48; T × C × V: Wilk’sλ=0.2568; F =16.09] were significantly different ( P <0.0001) with regard to all of the four quantitative traits. ANOVA indicated that the main effects of temperature, cultivar and variate, as well as all interactions, were significant for all the traits of study ( Table 1). The mean values and standard deviations for the observed traits indicated a high variability among the tested combinations, for which significant differences were found in terms of all the analyzed traits ( Table 2).

After 48 hours, the specimens immersed in the tomato root diffusate and in water retained their

Source d.f. Immobile J 2 Immobile J 2 Immobile J 2 Immobile J 2

of variation after 24 h after 48 h immersed after NaOH

in water treatment Temperature ( T) 2 8723.15*** 38277.8*** 943.39*** 938.374***

Cultivar ( C) 1 40703.81*** 8983.6*** 257.202*** 262.371*** Variant ( V) 5 9691.42*** 34681.6*** 583.498*** 582.31***

TC 2 159.67* 3219.1*** 47.557** 47.016**

TV 10 3228.36*** 7990.8*** 440.458*** 441.152*** CV 5 5546.58*** 2276.7*** 184.671*** 183.297*** TCV 10 2071.8*** 884.7*** 104.192*** 104.979***

Residual 396 45.1 120.1 7.319 7.377

* P <0.05; ** P <0.01; *** P <0.001; d.f. – the number of degrees of freedom.

motility. Differences in the effect of seed diffusates of the examined common vetch cultivars on the motility of J 2 forms were observed. Ina cultivar seed diffusates (with a high content of cyanogenic compounds) influenced the behaviour of J 2 stage through limiting their ability to move and caused paralysis at each of the tested temperatures, regardless of whether they were subjected to the sterilization process ( Table 2).

In the case of the Jaga cultivar (low content of cyanogenic compounds), J 2 specimens became immobilized (paralyzed) when placed in seed diffusates prepared in the tomato root filtrate after 48 hours of immersion at 17° C and at 21° C, regardless of the duration of exposure. A complete loss of motility was observed in J2 forms of the nematode exposed to the diffusates from sterile common vetch (cv. Ina), prepared in tomato root filtrate, incubated at 21° C. When subjected to the diffusates of common vetch (cv. Jaga) seeds, 100% of J 2 juveniles lost the ability to move after 48 hours of incubation at 21° C ( Table 2) .

After being transferred into Petri dishes with water, J 2 regained the ability to move (the paralysis subsided) at a rate of 90%. The mortality rate was 29% at the maximum, with the highest rate observed in the experiment variant using sterile seeds of the Ina cultivar, at a temperature of 21° C ( Table 2).

The correlation analysis performed indicated statistically significant correlation coefficients between immobile J 2 after 24 h and immobile J 2 after 48 h ( R =0.7419), as well as immobile J 2 immersed in water and immobile J 2 after NaOH treatment ( R =1.000) ( Table 3).

In the presented dendrogram, as a result of the nearest neighbour grouping using the Euclidean distances method, all the examined combinations were divided into three groups ( Fig. 1 ). The first group comprised four combinations (29, 30, 35 and 36), and the second group contained only one combination (27).

Individual traits are of different importance and have a different share in the joint multivariate variation. A study on the multivariate variation for combinations also includes identification of the most important traits in the multivariate variation of combinations. Analysis of canonical variables is a statistical tool that makes it possible to solve the problem of multivariate relationships ( Seidler-Łożykowska et al., 2013; Lahuta et al., 2018; Wrońska-Pilarek et al., 2018; Bocianowski and Majchrzak, 2019). Figure 2 shows the variability of the four traits of 36 studied combinations of cultivars, temperatures, and variants in terms of the first two canonical variables. In the graph, the coordinates of the point for particular combinations are the values for the first and second canonical variables, respectively. The first two canonical variables accounted for 80.8% of the total multivariate variability between the individual combinations ( Fig. 2 ). The most significant, positive, linear relationship between the first canonical variables was found for immobile J 2 after 24 h ( R =0.9737, P <0.001) and immobile J 2 after 48 h ( R =0.8736, P <0.001). The second canonical variable was significantly negatively correlated with immobile J 2 immersed in water ( R =-0.91, P <0.001) and immobile J 2 after NaOH treatment ( R =-0.9102, P <0.001). The greatest variation in terms of all the traits jointly (measured Mahalanobis distances) was found for combinations 27 (21° C, Cultivar 1, J 2+StS+water) and 30 (21° C, Cultivar 1, J 2+StS+ RD)

Combination Temperature Cultivar Variant Immobile J 2 Immobile J 2 Immobile J 2 Immobile J 2 after 24 h after 48 h immersed in after NaOH water treatment Mean ± S.d. Mean ± S.d. Mean ± S.d. Mean ± S.d.

1 10° C Ina J 2+ H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 0 ± 0 f

2 10° C Ina J2 + S + H 2 O 34.72 ± 13.062 de 29.44 ± 12.936 hi 2.778 ± 1.297 ef 2.778 ± 1.297 de

3 10° C Ina J2 +StS+ H 2 O 43.33 ± 12.713 c 30.56 ± 15.031 ghi 1.111 ± 2.171 fg 1.111 ± 2.171 ef

4 10° C Ina J 2+RD 0 ± 0 i 0 ± 0 k 0 ± 0 g 0 ± 0 f

5 10° C Ina J 2+ S +RD 8.06 ± 4.597 h 22.22 ± 13.731 ij 1.389 ± 1.716 fg 1.667 ± 2.247 ef

6 10° C Ina J 2+StS+RD 20 ± 9.535 f 34.44 ± 8.165 fgh 1.944 ± 1.716 fg 1.944 ± 1.716 ef

7 10° C Jaga J 2+ H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 8 10° C Jaga J 2+ S + H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 9 10° C Jaga J 2+StS+ H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 10 10° C Jaga J 2+RD 0 ± 0 i 0 ± 0 k 0 ± 0 g

11 10° C Jaga J 2+ S +RD 0 ± 0 i 0 ± 0 k 0 ± 0 g

12 10° C Jaga J 2+StS+RD 0 ± 0 i 0 ± 0 k 0 ± 0 g

13 17° C Ina J 2+ H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g

14 17° C Ina J2 + S + H 2 O 18.33 ± 10.2 fg 14.72 ± 19.667 j 0 ± 0 g

15 17° C Ina J2 +StS + H 2 O 21.94 ± 15.6 f 24.72 ± 15.793 i 4.167 ± 4.949 de 16 17 ° C Ina J2 +RD 0 ± 0 i 0 ± 0 k 0 ± 0 g

17 17° C Ina J 2+ S +RD 36.39 ± 10.489 d 48.89 ± 15.33 d 0 ± 0 g

18 17° C Ina J 2+StS+ RD 36.11 ± 9.83 d 38.89 ± 14.094 efg 1.667 ± 1.741 fg

0 ± 0 f

0 ± 0 f

0 ± 0 f

0 ± 0 f

0 ± 0 f

0 ± 0 f

0 ± 0 f

0 ± 0 f

4.167 ± 4.949 d

0 ± 0 f

0 ± 0 f 1.667 ± 1.741ef

19 17° C Jaga J 2+ H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 0 ± 0 f

20 17° C Jaga J 2+ S + H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 0 ± 0 f

21 17° C Jaga J 2+StS+ H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 0 ± 0 f

22 17° C Jaga J 2+RD 0 ± 0 i 0 ± 0 k 0 ± 0 g 0 ± 0 f

(COnTinUed)

Combination Temperature Cultivar Variant

23 17° C Jaga J 2+ S + RD

Immobile J 2 Immobile J 2 Immobile J 2 Immobile J 2 after 24 h after 48 h immersed in after NaOH water treatment Mean ± S.d. Mean ± S.d. Mean ± S.d. Mean ± S.d. 0 ± 0 i 45.83 ± 14.848 de 1.667 ± 2.247 fg 1.667 ± 2.247 ef

24 17° C Jaga J 2+StS+RD 0 ± 0 i 39.17 ± 14.293 efg 0.556 ± 1.297 g

25 21° C Ina J2 + H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g 26 21° C Ina J2 + S + H 2 O 17.78 ± 10.184 fg 39.72 ± 11.322 ef 7.778 ± 4.103 c 27 21° C Ina J2 +StS+ H 2 O 29.44 ± 9.727 e 38.33 ± 9.924 efg 29.167 ± 10.648 a 28 21° C Ina J2 +RD 0 ± 0 i 0 ± 0 k 0 ± 0 g

29 21° C Ina J 2+ S +RD 53.61 ± 14.387 b 71.11 ± 22.398 c 1.389 ± 1.716 fg 30 21° C Ina J 2+StS+RD 100 ± 0 a 100 ± 0 a 0.556 ± 1.297 g

31 21° C Jaga J 2+ H 2 O 0 ± 0 i 0 ± 0 k 0 ± 0 g

32 21° C Jaga J2 + S + H 2 O 14.44 ± 7.566 g 44.17 ± 15.707 de 6.944 ± 3.001 cd 33 21° C Jaga J2 +StS+ H 2 O 16.67 ± 6.816 fg 15.56 ± 7.698 j 10.556 ± 7.083 b 34 21° C Jaga J2 +RD 0 ± 0 i 0 ± 0 k 0 ± 0 g

35 21° C Jaga J 2+ S +RD 20.83 ± 6.376 f 100 ± 0 a 2.778 ± 3.978 ef

0.556 ± 1.297 f 0 ± 0 f

7.778 ± 4.103 c 29.167 ± 10.648 a 0 ± 0 f

1.389 ± 1.716 ef 0.556 ± 1.297 f

0 ± 0 f

6.944 ± 3.001 c 10.556 ± 7.083 b

0 ± 0 f

2.778 ± 3.978 de

36 21° C Jaga J2 +StS+RD 18.33 ± 8.704 fg 84.17 ± 24.168 b 1.667 ± 2.659 fg 1.667 ± 2.659 ef LSD0.05 5.39 8.795 2.171 2.18

* J 2+ H 2 O - juveniles of second stage immersed in water; J 2+ S + H 2 O - juveniles of second stage immersed in seeds diffusates of ViCia SaTiva in water; J 2+StS+ H 2 O - juveniles of second stage immersed in surface-sterilized seeds diffusates of ViCia SaTiva in water; J 2+RD - juveniles of second stage immersed in root diffusates of S. lyCOPeRSiCUm ; J 2+ S +RD - juveniles of second stage immersed in seeds diffusates of ViCia SaTiva in root diffusates of S. lyCOPeRSiCUm ; J 2+StS+RD - juveniles of second stage immersed in surface-sterilized seeds diffusates of ViCia SaTiva in root diffusates of S. lyCOPeRSiCUm . Means followed by the same letter are not statistically different.

Trait Immobile J 2 after 24 h

Immobile J 2 after 24 h 1 Iimmobile J 2 after 48 h 0.7419*** Immobile J 2 immersed in water 0.2069

Immobile J 2 Immobile J 2 Immobile J 2 after 48 h immersed after NaOH in water treatment

1

0.2241 1

Immobile J 2 after NaOH

0.2066 0.2241 1*** 1 treatment

*** P <0.001.

Effect of Vicia sativa on J 2 Meloidogyne hapla : Dobosz et al.

Hsp family Gene C. elegaNS Gene M. Hapla Gene location on (Transcript ID) (Transcript ID) contig

Hsp90 HSP 90 ( C47 E8.5) MhA1_Contig1972.frz3.gene3 4732..7425 Hsp70 HSP 1 ( F26 D10.3) MhA1_Contig113.frz3.gene45 79493..86067 Hsp60 HSP 60 ( Y22 D7 AL.5) MhA1_Contig737.frz3.gene23 48523..50607

sHsps HSP 43 ( C14 F11.5 ) MhA1_Contig30.frz3.gene18 50262..54876 sHsps hsp12.3 ( F38 E11.1) MhA1_Contig199.frz3.gene23 35708..36080

(the Mahalanobis distance between them amounted to 16.379). The greatest similarity (0.00) was found for 18 combinations.

Figures

Figure 1: Dendrogram of the nearest neighbour cluster grouping of combinations of temperature, cultivars, and variants on the basis of four traits.

Figure 2: Distribution of 36 combinations of temperatures, cultivars, and variants in the space of the first two canonical variables.