Sensitivity to salinity at the emergence and seedling stages of barnyardgrass (EchinochloQ crus-gQlli), weedy rice (OryzQ sQtivQ), and rice with different tolerances to ALS-inhibiting herbicides Author Fogliatto, Silvia Postdoc Fellow, Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università degli Studi di Torino, Grugliasco, Author Patrucco, Lorenzo Postgraduate Fellow, Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università degli Studi di Author Milan, Marco Postdoc Fellow, Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università degli Studi di Torino, Grugliasco, Author Vidotto, Francesco Associate Professor, Dipartimento di Scienze Agrarie, Forestali e Alimentari, text Weed Science 2021 Cambridge, England 2020-10-12 69 1 39 51 http://dx.doi.org/10.1017/wsc.2020.72 journal article 298657 10.1017/wsc.2020.72 f11a544c-edfb-4ceb-8f04-b1cee3d7a0ad 1550-2759 11553267 Oryza sativa (Weedy Rice and Rice) Seedling Development Plant Emergence. Seedling emergence of O. sativa and rice was affected more by salinity than E . crus-galli emergence was. Plant emergence for 0 mM NaCl was quite similar for all the O. sativa populations and Baldo, with values of about 90% or above; only CL 80, the variety tolerant to imazamox, displayed values of about 60% ( Figure 2 ). The comparison of parameter d of the curve showed an initial different emergence among all rice and O. sativa populations, including Baldo, and the CL 80 resistant variety ( Table 4 ). Slope was instead more similar between curves, and the only detected differences were between CL 80 and both populations wr2 and wr3. The point of inflection e of the curves was different between populations, as differences were found between population wr1 and all the other populations and rice varieties and between wr3 and Baldo. The most similar curves, showing nonsignificant differences between parameters and SI , were wr2 versus wr3 and wr2 versus Baldo ( Table 4 ). Emergence was quite similar to that of the control for 50 mM NaCl. A more marked emergence reduction occurred for 100 mM compared with the control, particularly in the CL 80 variety, which showed a 30% decrease in emergence, and an even more marked decrease occurred for 150 mM. Population wr1 displayed the highest emergence percentage for 200 mM, with an average emergence value of about 43%, while the lowest rate of emergence was recorded for Baldo (about 17%), CL 80, and wr2, with about 19% of germination, which corresponds to a reduction of about 80% compared with the control. All the O. sativa populations and rice varieties showed an emergence reduction of at least 90% for the highest NaCl concentration, compared with the control. A slightly higher emergence was recorded in the Baldo variety (8% emergence) for this salt rate, while emergence did not occur in the wr2 O. sativa population. Table 3. Chlorophyll a , chlorophyll b , chlorophyll a / b ratio, and carotenoid content of the different Echinochloa crus-galli populations and the percentage difference of these values for each salt concentration compared with the control.a
NaCl Chl a difference compared with Chl b difference compared Chl a / b Carotenoid difference compared
Population concentration Chl a b the controlc Chl b b with the controlc ratiob Carotenoidb with the controlc
mM mgg−1 SW % mgg−1 SW % mgg−1 SW %
s1 0 0.92 a — 0.41 a — 3.59 d 0.15 a —
50 1.32 c 42.96 e 0.89 c 114.99 d 1.49 b 0.23 c 54.57 e
100 1.31 b 41.29 e 1.01 d 144.69 e 1.29 a 0.21 b 42.99 e
150 1.33 d 44.27 D 0.57 b 37.22 B 2.36 c 0.34 d 129.56 C
s2 0 1.25 a — 0.47 a — 2.65 c 0.31 b —
50 1.33 c 6.54 c 0.91 d 91.78 c 1.68 b 0.27 a −12.16 c
100 1.32 b 5.79 c 0.84 c 77.75 c 1.57 a 0.28 a −10.60 c
150 1.39 d 11.33 B 0.82 b 73.87 D 1.60 a 0.34 c 9.44 A
s3 0 1.29 a — 1.17 c — 1.11 a 0.19 a —
50 1.33 c 2.67 b 0.92 b −21.30 a 1.45 b 0.25 b 32.76 d
100 1.32 b 2.09 b 0.92 b −21.05 a 1.47 b 0.25 b 35.65 d
150 1.36 d 5.33 A 0.69 a −40.69 A 1.97 c 0.34 c 82.64 B
r1 0 1.14 a — 0.63 a — 2.13 d 0.28 c —
50 1.32 c 15.77 d 0.95 b 50.90 b 1.39 b 0.23 b −19.53 b
100 1.28 b 12.88 d 1.30 c 106.34 d 0.98 a 0.14 a −51.47 b
150 1.38 d 21.33 C 0.95 b 50.98 C 1.44 c 0.31 d −8.92 A
r2 0 1.31 c — 0.70 a — 1.88 c 0.33 c —
50 1.26 a −3.46 a 1.49 c 113.51 d 0.85 a 0.06 a −80.39 a
100 1.30 b −0.41 a 1.14 b 63.01 b 1.15 b 0.20 b −39.36 a
150 — — — — — — —
aAbbreviations: Chl, chlorophyll; SW, shoot fresh weight. bLowercase letters refer to the comparison of the E . crus-galli chlorophyll a , chlorophyll b , chlorophyll a / b ratio, and carotenoid content for the NaCl concentrations within the same population. cThe E . crus-galli chlorophyll a , chlorophyll b , and carotenoid content variations among populations are indicated in lowercase letters in italics, lowercase letters in bold, and uppercase letters for 50, 100,and 150 mM concentrations, respectively.Different letters indicate significantly different values according to Tukey’ s honest significant difference test. Where no letters are present, the comparison was not significant. Table 4. Curve parameter (Equation 1) and sensitivity index (SI) (Equation 2) comparison between Oryza sativa populations/varieties (estimate and P-values).
Parameter b comparison Parameter d comparison Parameter e comparison SI
Population comparison Estimate P-value Estimate P-value Estimate P-value Estimate P-value
wr1/wr2 wr1/wr3 wr1/ CL 80 wr1/Baldo wr2/wr3 wr2/ CL 80 wr2/Baldo wr3/ CL 80 wr3/Baldo CL 80/Baldo 1.37 1.11 3.05 1.95 0.81 2.22 1.42 2.75 1.76 0.64 0.450 0.790 0.086 0.230 0.330 0.050* 0.330 0.028* 0.163 0.099 0.92 0.97 1.43 1.00 1.05 1.55 1.09 1.47 1.03 0.70 0.080 0.544 0.000* 0.997 0.268 0.000* 0.224 0.000* 0.656 0.000* 1.22 1.13 1.40 1.34 0.92 1.14 1.09 1.23 1.18 0.95 0.000* 0.008* 0.005* 0.000* 0.056 0.206 0.215 0.055 0.025* 0.683 1.22 1.13 0.71 0.748 0.926 0.87 0.91 0.81 0.85 1.05 0.000* 0.008* 0.000* 0.000 * 0.056 0.148 0.175 0.018* 0.008* 0.696
*Significant comparisons at P ≤ 0.05. Figure 2. Dose–response emergence curve with the average data points of the different Oryza sativa (weedy rice) populations and rice varieties against the salt concentration. Curve parameter estimates (Equation 1): wr1 ( b = 9.40, d = 87.14, e = 195.80), wr2 ( b = 9.40, d = 87.14, e = 160.19), wr3 ( b = 8.49, d = 89.73, e = 173.01), Baldo ( b = 4.81, d = 87.12, e = 146.49), CL80 ( b = 3.08, d = 60.89, e = 140.04). The salt concentration required to reduce the emergence by 50% (EC50) and the significant pairwise comparisons between EC50 (SI index) are shown below the graph (Equation 2). The NaCl concentration required to reduce emergence by 50% ( EC 50) was between 140 mM for CL 80 and 195 mM for the wr1 population. A pairwise comparison between EC50 values ( SI index) showed that wr1 had a significantly higher EC50 than all the tested O. sativa populations and rice varieties, thus suggesting a higher tolerance to salt. Moreover, both rice varieties showed a significantly lower value of EC50 than the wr3 O. sativa population. Oryza sativa and rice showed a higher sensitivity to salt than E . crus-galli , as demonstrated by the lower EC50 values. Moreover, similar values for emergence reduction were recorded for a salt concentration of 250 mM in E . crus-galli , but for lower concentrations in rice and O. sativa , that is, for values between 150 and 200 mM ( Figures 1 and 2 ). Previous studies also demonstrated that a moderate level of soil salinity (between 40 and 100 mM) still permits rice to grow, but some weeds, such as E . crus-galli , can germinate and even grow better than rice under these conditions ( Chauhan and Johnson 2009 ). Another study graded O. sativa as moderately tolerant to salinity, while E . crus-galli was classified within the salt-tolerant species, as its germination was about 30% for salt concentrations of 240 mM ( Hakim et al. 2011 ). A previous study on the sensitivity of Italian O. sativa populations and rice varieties to salt showed that CL 80 was affected more by salt, in terms of germination, than other herbicide-sensitive O. sativa populations and the conventional Baldo rice variety, which was the least affected by salt ( Fogliatto et al. 2019 ). In this study, the Baldo variety was less tolerant to salt, in terms of emergence, compared with germination, as the wr1 O. sativa population showed a higher emergence rate for increasing salt levels. This was also demonstrated by the higher EC50 displayed by wr1 compared with all other O. sativa and rice varieties, including Baldo. The CL 80 variety was confirmed to be salt sensitive, even at emergence, as it showed the highest emergence reduction across all salt concentrations. A certain variability in salt tolerance among rice varieties was demonstrated in previous studies, and the Baldo variety was classified as moderately sensitive or tolerant to salt according to the varieties to which it was compared ( Bertazzini et al. 2018 ). Apart from the abovementioned study on germination, which highlighted a high level of sensitivity to saline conditions in the CL 80 variety, no information regarding the sensitivity of this variety to salt is available ( Fogliatto et al. 2019 ). Plant Height, Shoot, and Root Weight. Oryza sativa and rice were observed to be sensitive to salt, as they were only able to develop seedlings for a concentration of 50 mM NaCl. After emergence, seedlings were not able to survive at higher salt levels, thus demonstrating the high sensitivity of O. sativa to salt at early seedling stages compared with emergence ( Table 5 ). The difference in plant height between the control and 50 mM NaCl was only significant for the CL 80 variety, for which salt reduced the height by about 35% compared with the control ( Table 5 ). Population wr2 showed a slight reduction in height (about 3%) with salt, while all the other populations were slightly stimulated, with Baldo and wr3 recording the largest increases. The reduction in shoot weight due to salt was again only significant for CL 80 (about 66% reduction compared with the control). All the other populations and Baldo showed a more moderate weight reduction, while population wr3 did not display any shoot weight reduction compared with the control. Root weight was affected more by salt than shoot weight, as a significant root weight decrease was observed for both rice varieties and wr2. The highest root weight reduction was observed for CL 80 (about 51% reduction) and wr2 (about 48% reduction), while population wr3 was affected the least (about 8% reduction). In general, at the seedling stage, O. sativa population wr3 seemed to be able to better tolerate saline conditions when compared with the resistant CL 80, as it showed a significantly greater plant height and lower root and shoot weight reduction. Population wr3 also showed a lower root weight reduction compared with wr2, in contrast to what was observed for emergence, in which population wr1 was the most tolerant. As already observed for emergence, among all populations and varieties, CL 80 was the most sensitive to salt. Table 5. Plant height and shoot and root weight of the different Oryza sativa populations (weedy rice) and rice varieties and the percentage difference of these growth parameters for each salt concentration compared with the control.
NaCl Plant Plant height variation Shoot Shoot weight variation Root Root weight variation
Population concentration height compared with the controla weight compared with the controla weight compared with the controla
mM cm % g % g %
wr1 0 16.97 — 1.93 — 1.55 —
50 17.14 1.03 ab 1.56 −19.14 b 1.04 −33.05 ab
wr2 0 19.08 — 1.94 — 1.86 —
50 18.43 −3.41 ab 1.41 −27.32 ab 0.96* −48.57 a
wr3 0 18.09 — 1.51 — 0.73 —
50 20.25 11.95 b 1.51 0.00 b 0.67 −8.64 b
Baldo 0 20.04 — 2.72 — 2.92 —
50 23.23 15.92 b 2.06 −24.36 ab 1.80* −38.43 ab
CL 80 0 12.61 — 0.29 — 0.27 —
50 8.20* −34.97 a 0.10* −65.91 a 0.13* −51.23 a
aLowercase letters refer to the growth parameter variations among populations for 50 mM. Different letters indicate significantly different values, according to Tukey’ s honest significant difference ( HSD ) test. Where no letters are present, the comparison was not significant. *Significant differences of the growth parameters, according to Tukey’ s HSD test, between the two NaCl concentrations (0 mM and 50 mM) for each population/rice variety. A lower tolerance of rice to salinity, compared with E . crus-galli , was found in previous studies in which rice was not able to develop seedlings at a high salt level, as rice is more sensitive to salt at emergence and in the early growth stages ( Aslam et al. 1993 ; Chauhan et al. 2013 ; Korres et al. 2019 ). When comparing the differences in salt tolerance between rice and O. sativa , it was found that the latter required higher salt levels than rice to achieve a 50% reduction in dry weight, thus highlighting an advantage of the weed over the crop in saline environments ( Cirillo et al. 2018 ; Tanji and Kielen 2002 ). In our study, regarding growth parameters, we found more variable behavior: CL 80 was found to be more sensitive than the O. sativa population wr3 and Baldo, which showed an intermediate tolerance, and was similar to that of some O. sativa populations. Plant height of rice and O. sativa was not significantly reduced by salt, except for CL 80, which appeared to be highly sensitive to salinity. Previous studies conducted on different rice varieties with low salt concentrations showed either a reduction in plant height for the most sensitive varieties or no change in plant height in the case of more tolerant varieties, thus highlighting a wide variability among genotypes ( Bertazzini et al. 2018 ; Hakim et al. 2014a ; Motamed et al. 2008 ). In our study, we confirmed both results, as only the most salt-sensitive variety showed a height decrease. The shoot and root weight were reduced more in the presence of salt, and this can be attributed to reduced photosynthesis and turgor, salt toxicity, and a disruption in mineral nutrition ( Bertazzini et al. 2018 ; Hakim et al. 2014c ). Some studies found that root and shoot weight were reduced by salt to a similar extent; in our study, we found a greater reduction in root weight. A previous study found that the rate of Na þ accumulation in rice was higher in roots than in shoots, and this resulted in a reduction in root biomass ( Hakim et al. 2014c ). Chlorophyll and Carotenoid Contents Chlorophyll content showed variable behavior in the presence of salt, depending on O. sativa population and rice variety (Table 6). In fact, chlorophyll a content increased at 50 mM in populations wr2, wr3, and Baldo compared with the control, while it was reduced at a greater extent in wr1 and CL 80. The wr1 O. sativa population showed the greatest reduction in chlorophyll, about 80%, followed by CL 80, which recorded a reduction of about 50%. Baldo showed the greatest increase in the presence of salt (about 68%). A similar trend was noted for chlorophyll b content for all populations and rice varieties, with reduced or increased values similar to those for chlorophyll a ; the only exception was population wr3, which had a reduction (about 16% decrease) in chlorophyll b content. Carotenoid content decreased to a great extent in population wr1, that is, a reduction of more than 70% was observed in the presence of salt, and this was followed by CL 80, with a reduction above 67%. Carotenoid content in populations wr2, wr3, and Baldo increased as salinity increased, with the highest value being recorded for Baldo (about 78% increase, compared with the control). The higher sensitivity to saline conditions of O. sativa and rice compared with E . crus-galli was also confirmed by the chlorophyll and carotenoid contents, which often showed a decrease in the presence of salt, in particular in the most sensitive O. sativa population (wr1) and rice variety ( CL 80); similar results were found in a previous study that showed a greater reduction of photosynthetic pigments in O. sativa than in E . crus-galli ( Hakim et al. 2014b ) . Concerning the growth parameters (plant height, shoot and root weight), CL 80 was more sensitive to salt than all other O. sativa and rice varieties; this sensitivity was also confirmed by the low photosynthetic pigment content. However, population wr1, which showed an intermediate sensitivity to salt in the growth parameters, recorded the highest chlorophyll and carotenoid content reduction, thus highlighting a greater sensitivity, which would probably also have been observed in seedling growth if the salt conditions had lasted longer. Baldo and wr3 were once again confirmed to be the most tolerant to salt when photosynthetic pigments were considered. The chlorophyll a / b ratio rose for increasing salt concentrations in all O. sativa populations and Baldo, while it did not vary at 50 mM compared with 0 mM in CL 80 (Table 6); as a high ratio is an indicator of a healthy plant, this confirmed the higher salt sensitivity of CL 80 ( Duarte et al. 2013 ). Even though population wr1 displayed the greatest pigment reduction, it also recorded a high chlorophyll a / b ratio; this behavior could probably be explained by the fact that during the degradation of chlorophyll b due to salt, to prevent further damage, chlorophyll b may be converted to chlorophyll a , which is the primary photosynthetic pigment ( Ashraf and Harris, 2013 ). The increase or moderate decrease in the chlorophyll content in salt-tolerant rice varieties observed in this study, as opposed to a marked decrease for salt-sensitive rice varieties, was also found in previous studies, thereby highlighting a high variability in salt response within Oryza sativa species ( Kibria et al. 2017 ). This study has highlighted a different tolerance to salinity of the tested species; E . crus-galli was found to be more tolerant than both O. sativa and rice. The level of tolerance varied according to the considered growth stage, and it was higher at emergence and lower at the seedling stage. The two weed species and rice were able to emerge up to 250 mM NaCl, while E . crus-galli was only able to grow for concentrations of up to 150 mM NaCl at the seedling stage, while rice and O. sativa only developed seedlings up to 50 mM. Previous studies found that salinity tolerance at the seedling stage was not correlated with salinity tolerance at other growth stages in rice ( Ferdose et al. 2009 ; Kakar et al. 2019 ). A further demonstration of this finding is the behavior of population wr1, which was the most salt tolerant at emergence, but not at the seedling stage. In some cases, relatively low concentrations of salt can stimulate plant growth, acting as a hormetic effect ( Calabrese 2013 ). In our study, hormesis was observed in E . crus-galli emergence and in the plant height of almost all populations for 50 mM NaCl. This phenomenon was less evident in O. sativa and rice, even though it was observed in some populations for 50 mM in terms of emergence (wr1 and wr2) and plant height (wr1, wr2, and Baldo). This phenomenon was reported in previous studies; in China , for example, some weed species were stimulated to germinate in soil taken from coastal areas with a moderate level of salinity ( Bai et al. 2014 ). Wide variability in response to salt was not only found for the different species but also among populations within the same species. The tested populations of both E. crus-galli and O. sativa responded variably to salt, in terms of growth parameters. Baldo and CL 80 demonstrated different salt tolerance, as CL 80 was observed to be more sensitive than Baldo. Wide growth variability has been demonstrated for both E . crus-galli and O. sativa , even under nonsaline conditions, as weeds are generally characterized by wide phenotypic plasticity, as well as genetic variability, in response to different environmental conditions ( Chauhan and Johnson 2009 ; Fogliatto et al. 2011 , 2012 ). This study also aimed to establish whether ALS inhibitor– resistant and ALS inhibitor–sensitive E . crus-galli populations and rice varieties responded differently to salinity. The ALS inhibitor–resistant E . crus-galli population r2 was affected more by salt, in terms of emergence, seedling growth, and carotenoid content, but this was less evident in the other resistant population r1; a behavior similar to that of r2 was observed for CL 80, which is tolerant to an ALS-inhibiting herbicide. Even though only a few resistant populations were tested, the study highlighted a common behavior in response to salt in two out of three herbicide-resistant populations (r2 population for E . crus-galli and CL 80); this behavior is probably caused by a fitness penalty that may be associated with herbicide resistance, even though specific studies are needed to confirm this hypothesis ( Keshtkar et al. 2019 ). Greater growth performance and higher competitive ability of herbicide-sensitive populations were also reported in a previous germination study on O. sativa and rice, while similar competitive ability was observed when comparing E . crus-galli populations resistant and sensitive to propanil and clomazone ( Bagavathiannan et al. 2011 ; Fogliatto et al. 2019 ). However, previous studies carried out with other species that are resistant to different herbicides found a higher growth performance and tolerance to salt in resistant populations ( Shrestha et al. 2018 ). A study conducted on junglerice [ Echinochloa colona ( L .) Link] found that glyphosate-resistant biotypes were more competitive, germinate better, and produce more biomass under moisture and saline conditions than sensitive populations ( Shrestha et al. 2018 ). Therefore, a lower growth performance of resistant populations in response to diverse environmental stresses cannot be generalized, and it may vary for different species, different populations within a species, and different herbicides and their related modes of action ( Shrestha et al. 2018 ).
NaCl Chl a variation compared with the Chl b variation compared with Chl a / b Carotenoid variation compared
Population concentration Chl a controlb Chl b the controlb ratio Carotenoid with the controlb
mM mg g− 1 SW % mg g−1 SW % mg g− 1 SW %
wr 1 0 1.10 — 0.20 — 5.48 1.13 —
50 0.21 * − 81.20 a 0.03 * − 85.45 a 7.08 * 0.31 * − 72.90 a
wr 2 0 0.96 — 0.17 — 5.68 1.02 —
50 1.22 * 26.67 d 0.27 * 56.92 d 4.58 * 1.28 * 25.26 d
wr 3 0 0.53 — 0.08 — 6.23 0.64 —
50 0.54 * 2.39 c 0.07 * − 16.66 c 7.67 * 0.78 * 21.13 c
Baldo 0 0.54 — 0.14 — 3.85 0.68 —
50 0.90 * 68.13 e 0.23 * 67.90 d 3.96 * 1.21 * 78.50 e
CL 80 0 0.97 — 0.12 — 8.28 0.78 —
50 0.48 * − 50.51 b 0.07 * − 43.70 b 7.27 0.25 * − 67.58 b
Table 6 . Chlorophyll a , chlorophyll b , chlorophyll a / b ratio, and carotenoid content of the different Oryza sativa (weedy rice) populations and rice varieties and the percentage difference of these values for each salt concentration compared with the control. a aAbbreviations: Chl , chlorophyll; SW, shoot fresh weight. bLowercase letters refer to the chlorophyll and carotenoid content variations among populations for 50 mM . Different letters indicate significantly different values according to Tukey’ s honest significant difference ( HSD ) test. Where no letters are present , the comparison was not significant. * Significant variations of the chlorophyll , chlorophyll a / b ratio , and carotenoid content, according to Tukey ’ s HSD test, between the two NaCl concentrations (0 mM and 50 mM) for each population / rice variety. This study highlighted that Baldo performed better under high salt concentrations than CL 80; thus the availability of salt-tolerant rice varieties is particularly important, as the majority of the rice areas throughout the world are located near coastal areas and river deltas where salinity problems are more common ( Formentin et al. 2018 ). Moreover, some weeds can be more competitive than others in saline environments ( Fogliatto et al. 2020 ); according to our study, E . crus-galli is favored more than O. sativa under saline conditions, and thus more efforts should be made to control this weed. Populations resistant to ALS inhibitors showed variable behavior, with some populations that tolerate salt (such as the E. crus-galli r1 population) and others that can be more sensitive to salt (such as the E. crus-galli population r2 and the CL 80 rice variety).