Pre-emergent indaziflam can enhance forest seed germination in direct seeding?

Abstract Pre-emergent herbicides can contribute to the control of weed competition in direct seeding restoration, however it is necessary to evaluate their effects on seeds of native tropical forest species. The aim of the study was to assess the potential impact of the herbicide indaziflam on the germination of 17 forest species. For this, a dosage of 180 mL of the product in 200L of water was compared to the control without herbicide. The degree of sensitivity of each species was calculated by a ratio between the percentage of germination with herbicide (GH) and the control without herbicide (GC) classifying them as: extremely sensitive (ES= (GH/GC) <0.25), sensitive (S=0.25< (GH/GC) <0.50), low sensitivity (LS=0.50< (GH/GC) <0.75), indifferent (I=0.75< (GH/GC) <1.0) and potentiated (P= (GH/GC) >1). The herbicide promoted a significant reduction in mean germination in 35% (n=6) of the species and 59% (n = 10) were sensitive or extremely sensitive to indaziflam, and only three did not germinate. On the other hand, 29.4% (n=5) showed low sensitivity or indifference to the herbicide, while seed germination was slightly increased by indaziflam to 11.7% (n=2). Pre-emergent indaziflam can be recommended in direct seeding restoration, as only 17.6% (n=3) of the species were inhibited by pre-emergent. However, the effect of indaziflam varies by species and requires further studies to support large-scale use in direct seeding.

In order to assess the selectivity of a pre-emergence herbicide in direct seeding restoration, it is necessary to use mathematical attributes to understand the dynamics of the seed germination process and to evaluate the quality of the seeds.One of the most important attributes is the germination percentage (G%) that allows identifying whether the seeds are capable of forming a healthy plant under favorable conditions (ISTA, 2022).Furthermore, the germination speed index (GSI) and the mean germination time (MTG) indicate the seed vigor expressed by the relationship between the number of germinated seeds and the germination time.In addition, the synchrony test measures germination synchrony (Z) and expresses the degree of germination overlap, with Z values equal to zero showing seeds that distribute germination over time (Ranal and Santana, 2006).
Pre-emergent herbicide is an effective method to control weed and invasive species (Holfus et al., 2021), however can cause injury or adverse effects on emergent seedlings (Davies et al., 2014).The use of herbicides must avoid damage to the germination process or to the soil seed bank, reducing losses in the emergence phase and ensuring the establishment of post-emergence plants.Indaziflam is a broad-spectrum pre-emergent approved for forestry, restoration and other cultures to control invasive grasses (Sebastian et al., 2016) and it has been studied by Brazilian researchers, mainly in relation to long-term effects for perennial crops (Guerra et al., 2013).The herbicide indaziflam has a long permanence in the soil, and as the sown seeds can be exposed to the herbicide for up to 180 days, we hypothesize that it can interfere with the seed germination process, regardless of the sown species.Due to the relevance of weed control for the restoration by direct seeding, the present study aimed to evaluate the potential impact of the indaziflam on the seed germination of tropical forest native species.

Material and Methods
Native forest species used in restoration by direct seeding were selected, based on our previous field experience (Table 1).We evaluated the resistance of the species to the pre-emergent Esplanade® with the active ingredient indaziflam, adopting a dosage of 180 mL of the product (20% above recommended by the package leaflet) in 200L of water compared to a control without herbicide.Seed lots were purchased from suppliers recently stored and evaluated according to the general methods of the International Seed Testing Association (ISTA, 2022).We determined the number of seeds.kg - and the mass of 1,000 seeds by purity test, and assessed seed germination with four replicates of 25 seeds per species by treatments without (control-C) and with indaziflam (herbicide-H), and we adopted dormancyovercoming when necessary (Table 1).
In the germination tests of larger seeds, we used as substrate two sheets of filter paper pre-moistened with 5mL of distilled water (control-without indaziflam) or 5mL of the herbicide solution.Smaller seeds (<100,000 seeds/kg)

Introduction
Direct seeding is a viable option in ecological restoration programs, as it allows greater control of the species composition, richness and plant density in restored sites (Stanturf et al., 2014).Besides, it also reduces the costs, nursery operations for the seedlings production, the use of equipment and permits minimum operational plans (Grossnickle and Ivetić, 2017).Many areas for restoration by direct sowing are abandoned or degraded pastures dominated by fast-growing exotic grass species, such as Urochloa spp.which inhibit the forest species growing and the natural regeneration (Resende and Leles, 2017).
The initial emergence in direct seeding depends on seed density and quality, and losses are concentrated in the emergence phase, occurring mainly up to 90 to 180 days after sowing (Ferreira et al., 2009).Seed density is around 250,000 to 500,000 seeds per hectare (Campos-Filho et al., 2013), however, seedling establishment in the field is low, ranging from 1,400 to 13,000 plants per hectare (Meli et al., 2018).Increasing the efficiency of direct seeding restoration, therefore, is directly linked to soil preparation and the control of post-emergence factors, such as weed control (Silva and Vieira, 2017).Even management practices such as manual or mechanized weeding can cause physical damage to young seedlings (Khaliq et al., 2013), making it necessary to seek management alternatives in the pre-and post-emergent phase.
Invasive species control is one of the most important practices in ecological restoration since weed species can hinder the development of planted species (Prior et al., 2018) and interfere in the establishment of plant community structure and assembly (Weidlich et al., 2020).The use of herbicides is the most used practice to control weed competition in restoration, but they can also impact regeneration or even the development of planted or sown species.
Herbicides are classified by selectivity (selective or non-selective for a particular crop), time of application (pre-or post-emergent), translocation (contact or systemic) and mechanism of action (Silva et al., 2009).Pre and/or post-emergence herbicides are used in direct seeding of agricultural crops (Mann et al., 2007;Khaliq et al., 2011), whose success depends on the dosage and persistence of the product in the field.The residual effect of a herbicide is related to its composition, which prevents the development of competition with weeds and, at the same time, does not cause damage to the soil seed bank or plants of more sensitive species (Dan et al., 2012).
The herbicide indaziflam (Esplanade®, Bayer CropScience) has a low leaching in any type of soil (Guerra et al., 2016) and is a member of the chemical family called nitriles (HRAC Group L).It acts by inhibition of cellulose biosynthesis (Tompkins, 2010) interfering in the growth of roots in the newly germinated seedlings (Clark, 2019).The herbicide has low solubility in water L -1 (2010 mg L -1 ) and low Kow (2.0 at pH=2.0 at pH=2.0 and pH=9).In Brazil, the use of indaziflam is recent and restricted to agricultural crops and silviculture of exotic tree species, presenting a broad spectrum of action in the Brazilian Journal of Biology, 2023, vol. 83, e268716   3/8 Pre-emergent can enhance seed germination?= ΣCn i,2 /N, where Cn i,2 = n i (n i -1)/2 and N = Σ n i (Σ n i -1)/2, where: Cn i,2 = combination of seeds that germinated in time 'i', two by two; n i = number of seeds that germinated at time "i" (Ranal et al., 2009).
We evaluated the effect of the herbicide on the mean germination time (MGT) by the ratio between the number of days to start germination without (C MGT ) and with the herbicide (WH MTG ).Therefore, ratio values greater than 1.0 (C MGT /H MGT > 1) indicated that the herbicide delayed (D) germination, increasing the average time; if C MGT /H MGT =1, the species was indifferent (I) to the herbicide or showed accelerated (A) (0<(C MTG /H MTG )<1), or inhibited (IH) (C MGT /H MGT = 0) germination in response to the herbicide.To compare the grade of sensibility for each species, we calculated a ratio between the percentage of germination with herbicide (G H ) and the control without herbicide (G C ) and classified species as: In order to verify whether there was a phylogenetic response of the species in relation to germination as a function of the herbicide application, we used the taxonomic distinction index (Δ*) (Clarke and Warwick, were placed in 10 cm diameter petri dishes with 2.5 mL of water (control) or 2.5 mL of herbicide solution incorporated.Species whose recommendation was vermiculite (Brasil, 2013;ISTA, 2022), we filled 11x11x3.5 cm germination-type boxes (gerbox) to half of their height with a fine-grained substrate and moistened with 100mL of distilled water (control) or the same volume of herbicide solution.
The gerbox and petri dishes were placed in Mangelsdorftype germinators under the recommended temperature conditions for the species (Table 1).The evaluation time for the germination test followed the instructions for analysis of seeds of forest species (Brasil, 2013).Weekly, we evaluated germination based on the primary root protrusion with at least 2 mm.After seedling growth or seed death, we classified seed germination as high (G>50%), medium (50% ≥ G >20%) or low (G>20%), and performed a variance coefficient per species for each treatment.
We assessed herbicide selectivity by the germination percentage (G%), and the seed vigor by estimating: (a) germination speed index-GSI = Σ (ni/ti), in which: ni = number of seeds that germinated at time 'i'; ti = time after test installation; (b) mean germination time -MTG = (Σn i t i )/Σn i , in which: n i = number of germinated seed per day; t i = test duration time; (c) germination synchrony -Z Table 1.Species and families with the description of the conditions for the germination test following the recommendation of the manual Instructions for Analysis of Seeds of Forest Species (Brasil, 2013)   species demonstrated more sensibility to indaziflam than others (as shown in Table 3).
From the seventeen species tested, 59% (n = 10) were sensitive or extremely sensitive to indaziflam, but only three species did not germinate, probably due to the herbicide (as shown in Table 3).On the other hand, 29.4% (n = 5) demonstrated low sensitivity or indifference to the herbicide, while seed germination was slightly potentialized by indaziflam to Hymenaea courbaril and Pterogyne nitens (as shown in Table 3).
2001).We applied the index since phylogenetic metrics make it possible to identify the accumulation of phenotypic and genetic as well as phenological response at different taxonomic levels (Morelli et al., 2018).
The non-parametric Mann-Whitney test was applied to compare the effect of the herbicide for each studied variable plotted in a box-plot, both performed in R Program (R Development Core Team, 2021).The taxonomic distinction index was calculated using the PAST 4.05 program (Hammer et al., 2001).

Results
The herbicide promoted a significant reduction (W= 901; p= 7.699 e-10 ) in the average germination in more than 80% (n=15) of the species, with a coefficient of variation close (83%) to the control treatment (82%) (as shown in Table 2; see Figure 1).
The pre-emergent indaziflam reduced significatively (W=1109; p-value=1.543e-07 ) the speed of seed germination in 47.7% compared to the control (GSI H = 0.7; GSI C = 1.5) (as shown in Table 2), however affected the synchrony of germination significantly (W= 758; p-value=5.94e-12 ) in relation to control (Ē H = 0.3 bits; Ē C = 1.7 bits) (as shown in and MTG (see Table 2).At the same time, the indaziflam reduced the variation among species in the speed of germination (GSI) (see Figure 1) and synchronized germination in relation to the control (as shown in Table 2).It is important to note that the lower the index of synchrony, the more synchronized is the germination in relation to time (Ranal and Santana, 2006).In direct seed, to reduce the time of germination and synchronize seed germination can increase the probability of species establishment.
The seed field emergence in direct seeding ranges from the 30th to the 90th (Soares and Rodrigues, 2008) and, in some cases, arriving up to 150-180th day after sowing.So, although indaziflam is influencing seed germination of some species, it has reduced mean time of germination and synchronized emergence which can favor seedling establishment in direct seeding.
The exact mechanism of action of this herbicide is still not completely clarified.It is known that cell wall formation is inhibited, but the synthesis of polysaccharide polymers is not affected, and cell walls already completely formed are not affected by the active ingredient indaziflam (Griffin, 2005).However, indaziflam can potentially constrain the radicle development and root growth of newly germinated seedlings by preventing cellulose formation (Clark, 2019).As indaziflam is a new herbicide option for long-term control in natural areas, more studies should be done to understand the effect on different species, mainly its impact on forest seed emergence in restoration by direct seeding.Then, to understand the indaziflam impact on

Discussion
Among the most sensitive species, P. guajava, G. integrifolia, P. confertiflora and P. cattleianum stand out, which do not present physical dormancy and have a thinner tegument, which can influence herbicide absorption, reducing seed germination.As pre-emergent herbicides act directly on seeds, by inhibiting cell wall biosynthesis (Tompkins, 2010), indaziflam decreases growth by suppressing cellulose synthesis (Brabham et al., 2014), and so may reduce seed germination.
Tegument thickness is one of the most important factors to control water absorption in seeds (Souza and Marcos-Filho, 2001).For the species Hymenaea courbaril and Pterogyne nitens, which are considered large seeds (Souza and Válio, 2001;Pereira et al., 2013), they did not suffer a negative effect from indaziflam due to their size and the physical dormancy, as their seed tegument is thicker, making it difficult for the product to enter.Although Senegalia polyphylla is sensitive to glyphosate, imazapyr, sulfentrazone and metribuzin (Monquero et al., 2011), the species was indifferent to indaziflam, (0.75< S< 1.0) and exhibited high germination (G > 50%) developing seedlings in both the presence and absence of the herbicide, even with a significant reduction in germination caused by the herbicide (see Table 3).
Although most species reduced the mean germination time with indaziflam, such as P. parviflora, M. urundeuva, A. tibourbou, C. trichotoma and P. dubium (see Table 2), their average germination was inhibited by the herbicide, indicating a compensatory and opposite effect between %G  seed germination requires an extensive survey on traits related to the responses of the species.The botanical family Fabaceae is one of the largest assembly of tree species of Angiosperms, with 2948 representatives throughout Brazil (JBRJ, 2022).Therefore, it is usually abundant in restoration programs with good results in direct seeding, especially for larger seed species (Palma and Laurance, 2015).The taxonomic diversity index indicated that there is a phylogenetic relationship higher for seeds without herbicide than those treated with indaziflam.The initial high quality of the seeds was more associated with the traits of the phylogenetic relationship of each species than with the germination responses in relation to the herbicide, which depends on other non-controlled factors.Confirming that, the species with the highest germination were concentrated in the Fabaceae family, as P. contetiflora, A. colubrina, S. polyphylla and P. parviflora (see Table 2).Although this family had the highest number of species, the taxonomic diversity index is not affected by the number of species tested at each taxonomic level (Gorenstein, 2009).

Gallesia integrifolia
There is potential for the use of pre-emergent indaziflam in direct seeding restoration.Even at doses higher than those of exotic tree species, only the three natives Pterogine contertiflora, Gallesia integrifolia and Psidium guajava were inhibited by the pre-emergent.Despite a negative impact on seed germination of some species, we identified a compensatory effect reducing the mean time of germination and an increase of synchronization, favoring concentration of seed emergence in a short period.However, the effect of indaziflam was variable by species and requires more studies to support a large-scale use on direct seeding.

Table 2 .
Germination percentage (G%) and standard deviation, mean germination time (MGT), germination speed index (GSI) and synchronization index (Ē) of the Seasonal Semideciduous Forest species tested with the pre-emergent herbicide indaziflam (H) and control (C).
Equal letters on the same line for each variable (%G and MGT) do not differ from each other by the Mann-Whitney test with p<0.05.CV = Coefficient of variation.Brazilian Journal of Biology, 2023, vol.83, e268716 5/8 Pre-emergent can enhance seed germination?

Table 3 .
Species with values for the grade of sensibility (S) and their classification(Class).and the values of the effect on the mean germination time (E MGT ) and their classification.