Allelopatic effects of Waltheria indica L . : Biocontrol potential in ecological

In Brazil, most degraded areas are occupied by exotic and invasive species, which require alternatives for their management. We evaluated the allelopathic effects of Waltheria indica in the laboratory from aqueous extracts of leaves and roots on the germination of the species Lactuca sativa L. (lettuce), Urochloa brizantha (Hochst. ex A.Rich.) RDWebster (brachiaria) and Handroanthus chrysotrichus (Mart. ex DC.) Mattos (yellow Ipe). We collected adult one-year-old Waltheria indica shrubs in ecological restoration areas by direct seeding. We used two treatments with aqueous extracts of leaves and roots and a control without extract, with ten repetitions of 10 seeds per treatment, totaling 600 seeds per test species. Germination and use of tetrazolium assays to evaluate the potential respiratory activity of the roots were used. The aqueous extract of W.indica leaves affected the number of germinated seeds of all test species, while the aqueous extract of roots affected only L. sativa and H. chrysotrichus. There were no significant differences between treatments (leaf and root extracts) in species germination. The aqueous extracts of Waltheria indica leaves and roots affected germination and cellular respiration of the studied species, mainly in Lactuca sativa. The identification of W. indica allelopathic compounds may be an initial step so that in the future new bioherbicides are produced from extracts of this species, or even that its seeds can be sown together with non-sensitive native species, aiming for control of exotic species in ecological restoration projects.


Introduction
Brazil is one of the five countries with the largest hotspot area in the world (Brancalion et al., 2019). Despite its importance, the country has high rates of deforestation, which has generated initiatives with targets to restore 12 million hectares of degraded areas and forests by 2030 (Calmon, 2021). However, most areas intended for ecological restoration are highly uncharacterized from the point of view of the composition of native species, represented mostly by degraded pastures (Rodrigues et al., 2009). Many of these areas to be restored have strong coverage of exotic and invasive plants that end up hindering the establishment of native species, becoming a major cause of failure in ecological restoration (Cornish & Burgin, 2005; García-Orth & Martinéz-Ramos, 2011).
Regardless of the occurrence and type of interactions caused by these exotic and invasive plant species, measures need to be taken to suppress or reduce the number of these plants in the area to be restored to the condition of coexistence with native species (Copeland et al., 2021). However, the control of invasive alien species is one of the main challenges for ecological restoration (Durigan et al., 2013;Holl et al., 2014;Weidlich et al., 2020). Currently, control can be achieved by applying preventive, biological, physical, and chemical methods, and integration between different methods is the most common and effective way to control exotic and invasive weeds (Silva et al., 2009;Brighenti and Oliveira, 2011;Passareti et al., 2020;Di Sacco et al., 2021).
Allelopathy is the process in which a plant produces chemical substances (allelochemicals) that can interfere with the growth and establishment of other plants (Lovett & Ryuntyu, 1992;Li et al., 2011). In this context, biological control by allelopathy, can be an alternative in the control of exotic invasive species, favoring ecological restoration (Lopes, 2017;Iqbal et al., 2020). Allelochemical substances can bring an ecological and sustainable alternative for the control of exotic and invasive species such as bioherbicides (Rosa et al., 2007;Rigon et al., 2013).
In areas of ecological restoration by direct seeding, we observed that, specifically around sites where there are densities of individuals of the native species Waltheria indica L. (white mallow), the litter layer was relatively continuous throughout the year. At these sites, there is a significant reduction in the occurrence of brachiaria grass (Urochloa spp.), considered an invasive exotic species; however, native species appear to be growing normally.
Based on these field observations, this study aimed to verify the allelopathic activity of aqueous extracts of leaves and

Material and Methods
Entire adult shrubs of Waltheria indica were collected in one-year-old direct seeding ecological restoration areas in the municipality of Cardoso, São Paulo State, Brazil (22 K 616245.75 m E 7781497.42 m S), on the Tomazão stream, a tributary of Turvo River that flows into Rio Grande River. The climate in the municipality of Cardoso is Aw by Köeppen (1948), a tropical climate with a dry winter season, with two well-defined seasons: a dry season, represented by a six-month period (April through September), and a very wet season, also of six months (October through March). The average temperature is 24.6 °C and the average annual rainfall is 1227 mm (Climate-date, 2021).
To evaluate the allelopathic effect of plant extracts on seed germination, two aqueous extracts were made, the first using adult leaves and the second using roots of Waltheria indica (white mallow). The leaves and roots were dried in an oven with forced air circulation at 45ºC (Melo et al., 2004), and after crushed. Deionized water was added at a temperature of 60º C to the dried and crushed material of each plant organ at a ratio of 2:10 (g/ml). The solutions were left to rest for 24 h for extraction of the compounds. After this period, the solutions obtained were strained through filter paper and stored in a refrigerator at 4ºC.
We selected as test-species the sensitive L. sativa cultivar aurelia to express the allelopathic effect, the invasive weed U, brizantha, and the native species H. chrysotrichus. We analyzed aqueous leaf and root extracts at the above mentioned concentrations in the germination bioassays. We utilized as control treatment, the filter paper moistened only with 6 ml of deionized water.
The germination tests were conducted in petri dishes with eight centimeters in diameter, lined with two layers of filter paper moistened with six ml of extract. Ten repetitions with 10 seeds each were used for each treatment (leaves, root and control), totaling 400 seeds per species. All treatments, discriminated by type of extract and control, were composed by 10 repetitions, were distributed entirely at random in a mangelsdorf-type germination chamber, at 25 ºC, under 12-hour lighting per day, maintained by four white fluorescent lamps. The germination test for lettuce seeds was conducted for 10 days and those of brachiaria and yellow Ipe for 15 days. At the end, the percentages of germination and the potential respiratory activity of the seedling's roots were evaluated. Seeds were considered germinated when they produced primary roots at least 2 mm long with geotropic curvature (Carmo et al., 2007).
To evaluate and estimate the potential respiration of the roots of the test-species seedlings, we used the tetrazolium test, which principle is the reduction of 2,3,5-tetrazolium triphenyl chloride through the activity of dehydrogenase enzymes, catalysts of respiratory reactions in cells, forming triphenylformazan, a red substance that indicates living tissues in which there is respiratory activity (França Neto, 1999).
Research, Society and Development, v.10, n.13, e235101321263, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21263 4 After pre-testing to define the most appropriate concentration and immersion time for staining, the roots were placed in black germination boxes containing tetrazolium solution (pH 6.5 to 7.0) at a concentration of 0.075% and placed in a 25 ºC B.O.D. germination chamber for 4 hours in the absence of light. At the end of the incubation period, the tetrazolium solution was discarded, and the roots were washed in running water, remaining moist and in the dark. Then, each root was examined individually with the aid of a stereoscopic magnifying glass, analyzing the extent of the colored tissues.
Percentages of germination and potential root respiratory activity were calculated. The Mann-Whitney test was used to check differences in germination between the treatments and their respective controls, after testing for normality and homogeneity of the data at 5% statistical significance. All analyses were performed in the program R (R Development Core Team 2021).

Results and Discussion
Allelopathic substances can be present and released by the stem, and especially by the leaves and roots (Piña-Rodrigues & Lopes, 2001;Kremer et al., 2016;Ribeiro et al., 2019). These substances can often be volatile and or affect only a few species (Rice, 1984). In addition, the allelopathic effect may affect some part of the plant's development, such as germination, growth, or establishment. (Putnam & Duke, 1978;Gatti, Perez & Ferreira, 2007). The aqueous extract of Waltheria indica leaves was found to affect the number of germinated seeds of all test species (Figure 1a), while the aqueous extract of W.
indica roots affected only L. sativa and H. chrysotrichus (Figure 1b). This may be associated with the fact that allelopathic substances affect species in different ways . Although the root extract did not directly affect the germination of the invasive weed U. brizantha, it was observed that its roots showed morphological differences in relation to the control and were shorter and thicker.  (Figure 2b). As already reported, the allelopathic effects can affect some part of the plant development (Putnam & Duke, 1978;Piña-Rodrigues & Lopes, 2001;Gatti, Perez & Ferreira, 2007;, and in the present study it was possible to verify, from the tetrazolium test, that even though the seeds germinated, the allelopathic substances affected the cellular respiration of the roots (Figure 3).  Source: the authors.
The toxic effects of the aqueous extracts of W. indica go beyond just inhibiting germination; they are associated with death of the seedling roots evaluated. Many studies with allelopathic compounds bring considerations about the growth and development of the tested species , and many species end up germinating; however, the development is affected, and they cannot become normal adult plants (Da Rocha et al., 2018;Bernardes et al., 2020;Fioresi et al., 2021).
There was a significant difference (p < 0.05) between the treatments and their respective controls only in the germination of L. sativa species in both extracts (Table 2). And for root respiration potential, significant difference (p<0.05) was found between treatments and their respective controls only for the roots of L. sativa species in both extracts (Table 3). All roots of U. brizantha and H. chysotrichus were dead in the bioassay with the aqueous extract of W. indica root.  There was no difference (p > 0.05) in germination of L. sativa seeds (W = 37.5, p = 0.3603) between aqueous extracts of leaves and aqueous extract of roots ( Figure 4A). The same was observed for the percentage of cellular respiration in L.
sativa roots between aqueous leaf extracts and aqueous root extracts ( Figure 4B). Since allelopathic compounds can be in all parts of the plant, especially in leaves and roots (Rice, 1984  Letter "a" shows that there was no difference between treatments at 5% significance using the Mann-Whitney test. Source: the authors.

Conclusion
The aqueous extracts of the leaves and roots of Waltheria indica affected the germination of the studied species, especially in Lactuca sativa. The absence of cellular respiration of seedling roots of the invasive U. brizantha reinforce the potential use of Waltheria indica to control weed, however it needs to be carefully examined since root extract also affected the native H. chrysotrichus. Therefore, we believed that the elucidation of the allelopathic compounds of W. indica may be an initial step for the study and development of bioherbicides to be produced in the future.