UVOD
Oak is one of the most common broadleaf forest trees in the northern hemisphere, represented by about 500 species worldwide (Bonner and Vazzo, 1987; Pang et al. 2019; Wang et al. 2022). In Türkiye, it is represented by 17 species (23 taxa in total) (Yılmaz, 2014). The General Directorate of Forestry's 2022 statistics reveal that oak is present on 6.8 million hectares of land, with 41% of high forest and 59% of coppice forest (OGM, 2023). In recent years, with the demand for increasing forest areas in oak species, the use of acorn material has also increased. Acorns cannot survive on the ground for long due to their recalcitrant seed characteristics. Even if buried under the soil surface by factors such as climatic conditions, wild animals, or people, they are still exposed to desiccation conditions during shedding (Tweddle et al. 2003). Species with high carbohydrate nutrient reserves, such as oak, lose moisture quickly (Bonner, 1996). Therefore, it is recommended to harvest acorns as soon as they physiologically mature (Bonner and Vazzo, 1987). Acorns are generally recalcitrant seeds and lose their ability to germinate when they lose moisture (Bonner and Vozzo 1987; Bonner 1996). Recalcitrant seeds are a short-lived class of seeds that should not fall below a certain moisture content and have a high moisture content when they mature or shed (Berjak et al. 1989; Suszka et al. 1996; Bonner, 2008; Boydak and Çalışkan, 2021). When the acorns are mature, white oaks contain ~50% moisture and red oaks contain ~40% moisture (Bonner and Vazzo, 1987). However, viability is lost when the moisture of the acorns decreases below 25-40%, depending on the species (Boydak and Çalışkan, 2021). The moisture content of acorns can vary depending on seasonal precipitation, evapotranspiration, and climatic conditions over the years (Tweddle et al. 2003). Seed desiccation damage is the main cause of this loss of viability (Finch-Savage, 1992). Loss of viability in recalcitrant seeds is a direct result of seed desiccation damage (Finch-Savage, 1992), and the sensitivity of these seeds to desiccation is so great that they can suffer fatal damage even at 5% moisture loss (Connor and Sowa, 2003; Bonner, 2008; Xia et al. 2012; Bewley et al. 2012). When the seeds begin to dry out, their viability somewhat decreases at first due to the decrease in moisture content, but then the viability significantly decreases at a certain moisture content. This is called “critical moisture content” or “lowest safe moisture content” (Fing-Savage, 1992; Hong et al. 1996). Viability is lost as desiccation time increases.
The study has three main objectives. Firstly, to determine the changes in moisture loss over time. Secondly, to investigate the effect of moisture loss due to desiccation on germination behaviour. And thirdly, to identify the highest moisture content and critical moisture content of acorns of Q. cerris, Q. petraea and Q. robur.
MATERIJALI I METODE
Study sites and acorn collections – Područje istraživanja i prikupljanja žira
Acorns were collected in October 2022 from natural distribution of Q. cerris, Q. petraea, and Q. robur in the Sinop province. The collection sites were Sinop-Central (410 57' 21" N, 340 48' 26" E; Karacaköy, 45 m a.s.l.) for Q. cerris, Sinop-Ayancık (410 56' 41" N, 340 44' 14" E; Ağaçlı village, 65 m a.s.l.) for Q. petraea, and Sinop-Central (410 57' 58" N, 350 05' 12" E; Çiftlik village, 30 m a.s.l.) for Q. robur. Acorns were collected from 10 mature trees of each of oak species, with a minimum distance of 50 m between them. Approximately 1500 acorns were collected from each oak species.
The collection sites exhibit the characteristics of the Black Sea climate in coastal areas and the continental climate in the inner regions. Based on climate data from the Turkish General Directorate of Meteorology between 1992 and 2022, the annual average precipitation is 735 mm and the annual average temperature is 14.8°C. Erinç (1965) defines precipitation efficiency as the ratio of annual average precipitation and annual average maximum temperature. According to Erinç (1965), Sinop has a precipitation efficiency index of 29.5, indicating a semi-humid climate.
Acorns were transferred to Sinop University Ayancık Vocational School Forestry Laboratory for measurement and laboratory testing. Acorns with abnormal discolouration and insect damage were removed. Only healthy acorns were used and identified through a flotation test (Bonner, 2003). For each species, 1500 seeds were stored in sealed polyethylene bags in a refrigerator at +2ºC until the tests were carried out.
Acorn morphological measurements and initial moisture content – Morfološka analiza žira i početni sadržaj vlage
Acorn length and width were measured using digital calipers (aogo) on 100 randomly selected acorns from each species. The weight of 1000 acorns was determined, and the moisture content was measured using 25 acorns (5 replicates of 5 acorns) for each species. The fresh weight (FW) of the acorns was recorded on a precision scale, and after they were dried in an oven at 105±2 °C for 18 hours, their dry weight was measured and moisture determinations were made (Bonner, 2008).
Desiccation treatments – Tretmani isušivanja
The highest and the lowest moisture content of the acorns were determined in two stages. The acorns were divided into sub-seed lots of 30 acorns in polyethylene bags. The highest moisture content of the seedlots was determined using precision scales, and the acorns were then stored in sealed bags in a refrigerator at +2ºC. The acorns' outer surface was sprayed repeatedly with distilled water until it was completely wet (about 10-15 ml per treatment). The weight of acorns was measured daily, and the process of spraying distilled water was repeated according to the established procedures (Finch-Savage, 1992; Suszka et al. 1996, Yılmaz, 2005). The bags were renewed once a week to prevent the spoilage of acorns by pathogens. The final moisture content of the acorns was determined when their weight remained constant, and the highest moisture content was found.
The second step was to determine critical moisture content. The acorns that reached the highest moisture content were left to desiccate at 30-35% relative humidity and at 15-18°C for 18 days. The weight of the acorns in each seedlot was then measured to determine the rate of acorn moisture loss as a function of time on the control day (D1), the 3rd day (D3), the 6th day (D6), the 9th day (D9), the 12th day (D12), the 15th day (D15) and the 18th day (D18) (Bonner, 1996; Ganatsas et al., 2017). Before performing the germination test on the acorn lots, they were dried in an oven at 105±2 °C for 16-18 hours and their moisture was determined. In the present study, the approximate moisture content reached with the germination percentage falling below 50% was determined as the critical moisture content (Bonner, 1996). The new moisture contents depending on the weight change of the sub-seed lots with known weight and moisture values were calculated according to the formulas below (Suszka et al. 1996).
MC1: initial moisture content (%), MC2: new moisture content (%), FW1: initial weight (gr), FW2: new weight (gr)
Germination tests – Ispitivanja klijavosti
Each germination test included 100 acorns of Q. cerris, Q. petraea and Q. robur (4 replicates x 25 acorns). Sterilized river sand in approximately 2 l plastic containers was used as a germination medium. Germination tests were carried out at a constant temperature of 20°C in the dark in a germination cabinet (NBİOTECH, NB-2050Q). The germinations were checked and recorded at regular intervals (Suszka et al. 1996). Acorns that had at least 5 mm radicles and showed geotropism were considered germinated (Caliskan, 2014). The germination tests were terminated after 28 days. The germination percentage and mean germination time (MGT) for each treatment were determined using the formula below (Bewley and Black, 1994).
MGT: mean germination time (day), t: number of days since the test started, n: number of acorns germinated that day
Statistical Analysis – Statistička analiza
The effects of changes in acorn moisture on germination percentage and mean germination time were determined using an ANOVA test. ANOVA for germination characteristics is based on the following model: Ylk =μ +Ml+ elk , where Ylk is the observed value. μ is the overall mean, Ml is the moisture content effect and elk is the error. Duncan’s post hoc test was applied to determine statistically significant differences in germination characteristics (α=0.05). Acorn moisture loss due to desiccation time was evaluated by linear regression analysis. SPSS 2021 software was used for statistical analysis.
REZULTATI
The average length, width, weight of 1000 acorns and moisture content of the acorns are given in Table 1. The highest moisture contents found amounted to 48.3% in Q. cerris, 51.1% in Q. petraea and 49.2% in Q. robur on D1 (Figure 1). The maximum moisture content was reached in 11 days for Q. cerris, 8 days for Q. petraea, and 10 days for Q. robur. Moisture levels in Q. cerris, Q. petraea, and Q. robur show a linear decrease after 18 days of desiccation. Acorns that were left to desiccate at their maximum moisture content reached their harvested moisture content after 3 to 6 days for Q. cerris, and after 1 to 3 days for Q. petraea and Q. robur (Figure 1). The effects of changes in the moisture contents of acorns in Q. cerris, Q. petraea and Q. robur on the germination percentage and mean germination time are given in Table 2.
Table 1. Morphometric characteristics of Quercus cerris, Quercus petraea, and Quercus robur acorns
Tablica 1. Morfometrijske karakteristike žira vrsta Quercus cerris, Quercus petraea i Quercus robur
*Moisture content of acorns when harvested. ± indicates the standard error.
Figure 1. Acorn moisture content loss of oak species ( Q. cerris, Q. petraea, Q. robur) at 15-18°C during 18 days of desiccation treatment
Slika 1. Gubitak vlage žira tri vrste hrasta ( Q. cerris, Q. petraea, Q. robur) na 15-18 °C tokom 18 dana tretmana isušivanja
Table 2. ANOVA results regarding the effect of moisture content loss in Q. cerris, Q. petraea, and Q. robur acorns on germination percentage (GP)
and mean germination time (MGT)
Tablica 2. Rezultati ANOVA testa koji prikazuju učinak gubitka sadržaja vlage žira Q. cerris, Q. petraea i Q. robur na postotak klijavosti i srednje vrijeme
klijanja
Figure 2. Effect of desiccation treatments on acorn germinations of Q. cerris, Q. petraea, and Q. robur
Slika 2. Učinak tretmana isušivanja na klijanje žira kod vrsta Q. cerris, Q. petraea i Q. robur
Table 3. The effect of acorn desiccation treatments on germination percentage and mean germination time in of Q. cerris, Q. petraea, and Q. robur
Tablica 3. Učinak tretmana isušivanja žira na postotak klijavosti i srednje vrijeme klijanja kod vrsta Q. cerris, Q. petraea i Q. robur
The means in the same column followed by the same letters are not statistically different in Duncan’s post hoc test (P<0.05); ± indicates the standard deviation.
Figure 3. Effect of desiccation treatments on mean germination time of acorns of Q. cerris, Q. petraea, and Q. robur
Slika 3. Učinak tretmana isušivanja na srednje vrijeme klijanja žira kod vrsta Q. cerris, Q. petraea i Q. robur
There were significant differences found in the desiccation treatments between the oak species in terms of GP and MGT. Decreases in germination percentages due to moisture loss in Q. cerris, Q. petraea and Q. robur are given in Figure 2. The effects of moisture content loss on MGT showed a similar pattern to the relationship between moisture content on germination percentage. The MGT increased as the moisture content decreased (Figure 3, Table 3).
Desiccation time had a notable impact on both the onset of germination and the overall germination percentage across all oak species, as shown in Figure 4. The initiation and completion of acorn germination shifted towards the final days of the germination test.
Figure 4. Acorn germinations of Q. cerris (A), Q. petraea (B), and Q. robur (C) as affected by desiccation treatments
Slika 4. Utjecaj tretmana isušivanja na klijanje žira kod vrsta Q. cerris, Q. petraea i Q. robur
RASPRAVA
In terms of acorn size, Q. robur has larger acorns than Q. cerris and Q. petraea. There is a positive correlation between acorn size and 1000 seed weight (Bonner, 2003). In the present study, 1000 seed weights of Q. robur and Q. cerris were greater than that of Q. petraea. Ganatsas et al. (2017) found that large and heavy acorns had lower moisture loss. Devetaković et al. (2019) stated that germination in the group of large acorns was more than twice as high as in the group of smaller acorns. Additionally, nursery studies have reported that using large acorns has a positive effect on seedling quality (Roth et al. 2009; Roth et al. 2011).
In the present study, Q. cerris, Q. petraea, and Q. robur had an average moisture content of 41-46% at the time of harvesting. In some studies, the initial moisture content at the time of the collection was determined as 35% in Q. petraea (Tilki, 2010), 33-35% in Q. ithaburensis (Ganatsas et al. 2017), and 44% in Q. ilex (Leon-Lobos and Ellis, 2018), and it was found that there were different values between species. The highest moisture content (D1) had the highest germination percentage in Q. cerris, Q. petraea, and Q. robur. It was reported that the germination performance of Q. robur seeds increased in the first days after being saturated (Özbingöl and O'reilly, 2005). On the other hand, Q. ithaburensis, acorn moisture content increased by 5.7%-6.9% in 24 hours (Ganatsas et al. 2017). In the present study, the highest water imbibition was observed in Q. cerris (17%), and then Q. petraea (11%), while the lowest water uptake was observed in Q. robur (9%). It was stated that the difference in water imbibition rates among species is due to the characteristics of the pericarp (Xia et al. 2012; Ganatsas et al. 2017).
In the present study, the acorn moisture contents of Q. cerris, Q. petraea, and Q. robur followed a similar linear pattern by decreasing with desiccation according to the days. The study results confirm that acorn behaviour against moisture loss is similar in acorn studies conducted by Suszka et al. (1996), Xia et al. (2012), and Chmielarz et al. (2022). The moisture content of Q. cerris, Q. petraea and Q. robur decreased by about 50% (48%-57%-55%) at the end of 18 days. It was reported that the reason for the change in acorn desiccation rates between and within the species was not related to acorn oil content and acorn weight, but this change was mostly related to the acorn surface area, volume and coat properties (Ganatsas and Tsakaldimi, 2013; Ganatsas et al. 2017). Q. petraea rapidly lost moisture and had lower germination percentage than Q. cerris and Q. robur. The loss of viability due to desiccation and the variation in moisture content may vary depending on the year of collection, origin and species (Leon-Lobos and Ellis, 2018). Differences in the rate of moisture loss may be due to the high moisture content of the acorn at the time of shedding and the characteristics of acorn coats of the species (Suszka et al. 1996, Xia et al. 2012, Ganatsas and Tsakaldimi, 2013). The germination percentage significantly decreased with moisture loss in Q. cerris, Q. petraea, and Q. robur (Figure 2, Table 3). While there were linear gradual decreases in moisture contents during the first 6 days of desiccation, the content of moisture loss increased from the 9th day. After the 12th day, the effect of desiccation significantly affected the germination percentages. It has been statistically observed that germination percentage is highly dependent on moisture content. The decrease in moisture content is associated with the loss of viability in acorns. Bonner and Vazzo (1987) and Suszka et al. (1996) reported that germination is reduced by half when the moisture content is below 30% and there is a complete loss of viability when the moisture content is below 25%. Similar results were found for Q. cerris, Q. petraea and Q. robur in the present study. In addition, even when the moisture content was less than 30%, germination of the acorns was observed, albeit at a low germination rate. These results can be explained by the presence of acorns with a high moisture content still in the batch, as well as by the presence of acorns with different sensitivity and different tolerance to desiccation (Finch-Savage, 1992; Xia et al., 2012). In the present study, as the moisture content decreased, germination percentage decreased and the MGT increased (Table 3). In different studies, the effect of decrease in moisture on MGT is similar (Connor and Sowa, 2003; Özbingöl and O'reilly, 2005, Ganatsas and Tsakaldimi, 2013). After 15 days of desiccation, MGT increased from 4.4 days to 10 days in Q. coccifera, from 2 days to 7.6 days in Q. pubescens and from 4.4 days to 6.8 days in Q. pedunculiflora. However, Q. pedunculiflora was less affected by moisture loss than Q. coccifera and Q. pubescens (Ganatsas and Tsakaldimi, 2013).
The moisture content of Q. cerris in groups D3 and D6 was close to the moisture content at harvest time (41%). Rapid loss of acorn moisture after D15 showed statistically rapid decrease in germination percentages. According to Leon-Lobos and Ellis (2018), desiccation of the moisture content from 47.5% to 31% in Q. cerris reduced the germination percentage from 93% to 50%, therefore suggesting that the safest moisture content is approximately 40%. Despite the decrease in moisture content (51%-45%) in Q. petraea in D1 and D3 groups, no significant differences were observed in the germination percentage and MGT. The greatest effect on acorn moisture loss was observed when it dropped from D12 to D15. According to Suszka et al., (1996), Q. petraea acorns had 50% or more moisture content at the time of shedding, and the critical moisture content was 40-42%. In Q. robur, there was no significant difference between moisture loss and germination percentages in D1 and D3 groups, but there was a significant difference between moisture loss and MGT. It can be stated that the effect of moisture loss after D12 was high. In the present study, Q. robur was at 27% moisture content in D18, the germination percentage was 36%, while according to Suszka et al. (1996), the germination percentage was 14% at the same moisture content. Besides, Suszka et al. (1996) suggested that the moisture content of Q. robur acorns should not fall below 40%. Finch-Savage (1992) reported that the viability of Q. robur acorns was completely lost at 19% moisture content, and Xia et al. (2012) reported that it could tolerate desiccation up to approximately 20% moisture content.
On the other hand, in the case of different oak species, Bonner (1996) stated that the critical moisture content of Q. nigra was 10-12%, while Tilki and Alptekin (2006) stated that the critical moisture content of Q. vulcanica was 16%. Connor and Sowa (2003) stated that the acorn viability of Q. alba decreased significantly when the acorn moisture dropped below 30%. In Q. fabri, when the acorn moisture content decreased from 46% to 22%, the germination percentage decreased from 95% to zero, and when the moisture content was 32%, the germination percentage was more than 50% (Tian and Tang, 2010). Ganatsas and Tsakaldimi (2013) stated that the critical moisture content was 26% during the desiccation period of 5-7 days in Q. coccifera and Q. pubescens. They determined that the germination rate of Q. ithaburensis subsp. macrolepis was 100% when the moisture content was 35%, and at the end of 15 days of desiccation, 25% germination occurred at 15% moisture content, while the limit moisture at which germination percentages started to decrease was 25%. Q. ithaburensis subsp. ithaburensis was not affected by desiccation at room temperature for 15 days (GP 97%).
ZAKLJUCAK
The critical moisture contents for Q. cerris, Q. petraea, and Q. robur were in the ranges of 28-31%, 31-36%, and 32-37%, respectively. Germination percentage significantly dropped if the moisture content fell below the critical level. For Q. cerris, the germination percentage decreased from 83% to 43% as the critical moisture content decreased. For Q. petraea, the germination percentage decreased from 100% to 44%, and for Q. robur, it decreased from 97% to 43%. One limitation of this study is that it only includes one region for Q. cerris, Q. petraea, and Q. robur species. More detailed results can be obtained by conducting studies on different populations of these three species. The present study provides guidance for nursery studies on Q. cerris, Q. petraea and Q. robur, from acorn harvest to post-seeding care.
Acknowledgments