extfieldsofstudy: Biology Medicine

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Sperm enrichment from poor semen samples by double density gradient centrifugation in combination with swim-up for IVF cycles

Sperm preparation in IVF cycles using density gradient centrifugation (DGC) in combination with swim-up (SU) has been widely adopted in reproductive centres worldwide. It is a fact that the sperm recovery rate following one DGC from poor semen samples (showing liquefaction defects/containing too many unresolvable clots or rare sperm) is relatively low. Our results showed that double DGC (DDGC) is effective at increasing the sperm recovery rate from poor semen samples. However, DDGC may increase the mechanical stress of sperm, thereby potentially impairing embryo development. Therefore, it is necessary to evaluate the safety of using sperm prepared by DDGC/SU for IVF cycles. In this study, we retrospectively analysed the data generated from a total of 529 IVF cycles (from June 2017 to June 2018), and these IVF cycles contributed 622 transfer cycles (from June 2017 to December 2018) in Changzhou Maternal and Child Health Care Hospital. Of them, 306 IVF cycles and the related 355 transfer cycles (normal semen samples prepared by DGC/SU) were set as the normal group, while 223 IVF cycles and the related 267 transfer cycles (poor semen prepared by DDGC/SU) were set as the observation group. The main outcome measures, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, clinical pregnancy rate and live birth rate, birth weight and duration of pregnancy, were compared between the two groups. Compared to semen in the DGC/SU group, semen in the DDGC/SU group showed increased levels of the DNA fragmentation index (DFI) and reduced sperm concentration, percentage of progressive motility (PR) sperm, and percentage of normal morphology sperm. The indicators reflecting in vitro embryo development and clinical outcomes were similar in the DGC/SU group and DDGC/SU group, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, pregnancy rate, implantation rate, spontaneous abortion rate, live birth rate, birth weight and duration of pregnancy. Furthermore, we found that the 1PN zygote formation rate was significantly lower in the DDGC/SU group than that in the DGC/SU group. We concluded that oocytes fertilized by sperm from poor semen samples separated by DDGC/SU achieved the same outcomes as oocytes fertilized by sperm from normal semen separated by DGC/SU, suggesting that DDGC/SU is an effective and safe method of sperm enrichment for poor semen samples in IVF. The main contribution of the present study is the verification of the effectiveness of DDGC/SU in improving sperm recovery from poor semen samples and the safety of using sperm prepared by DDGC/SU for IVF.

In in vitro fertilization (IVF), two major semen preparation methods have been widely used for selecting sperm from semen, namely density gradient centrifugation (DGC) and swim-up (SU). Numerous studies have focused on investigating which method is better for sperm enrichment. However, studies from different groups have drawn inconsistent conclusions [1][2][3][4][5][6] . Recent studies have suggested that DGC in combination with SU (DGC/SU) could be one of the most effective approaches 7,8 . Most Chinese reproductive centres, including ours, have adopted DGC/SU for enriching sperm in IVF.
In clinical sperm preparation, the sperm recovery rate following one DGC largely depends on semen quality. Generally speaking, the sperm recovery rate from poor semen samples (such as liquefaction defects, containing too many unresolvable clots or rare sperm) is far from satisfactory. In clinical settings, when purifying sperm from semen for conventional IVF, we often encounter the situation where only a small proportion of sperm from a poor semen sample was obtained after one DGC. We have to make a choice: ignore the remaining motile sperm in the semen or re-extract them. In our reproductive centre, if few sperm are obtained after one DGC, the sperm precipitation (visible or invisible) is collected, and the remaining semen undergoes a second round of DGC with the old gradient column. Actually, the sperm from the double consecutive DGC would be pooled together for the next swim-up procedure. Here, we defined the double consecutive DGC in combination with SU as DDGC/SU. It has been demonstrated that an increase in the centrifugation time or gravitational (g) force can increase the sperm recovery rate from equine semen but can also decrease sperm motility or quality due to the mechanical forces associated with centrifugation and excessive packing of the sperm 9 . Studies using spermatozoa from the epididymis of rats and mice revealed that increasing the centrifugation force decreased sperm function [10][11][12] . A previous study demonstrated that centrifugation stress reduces the responsiveness of the spermatozoa of pigs to a capacitation stimulus 13 . Although the sperm of bull are relatively resistant to high centrifugal forces, it has been demonstrated that a reduction in centrifugation force has increased the fertilization rate in vitro 14 . Studies investigating the influence of centrifugation on human sperm showed that centrifugation induced damage to human sperm 15,16 . Indeed, in the abovementioned studies, either conventional centrifugation or DGC was used for sperm preparation. Although it is believed that DGC causes less damage to sperm than conventional centrifugation, centrifugation per se is harmful 16 . Therefore, it raises the concern that mechanical stress induced by either the increased centrifugation force or additional centrifugation time will further increase sperm damage, which may potentially affect the process of fertilization or embryo development. However, studies correlating the mechanical stress of sperm with embryo development are lacking. Although it has been demonstrated that sperm processing by DGC and SU is effective at reducing sperm with DNA damage, which is thought to definitely adversely affect early embryo development, it remains elusive whether a second DGC will introduce new damage to sperm, thereby finally impairing embryo development 17,18 .
Although the sperm from both the first and second DGC were gathered, the safety of DDGC/SU for sperm needs to be evaluated. In this study, we retrospectively analysed the data generated from patients from June 2017 to December 2018 in the reproductive centre of Changzhou Maternal and Child Health Care Hospital for IVF treatment. According to the method of sperm processing, patients were divided into two groups: the DGC/SU group and the DDGC/SU group. The data generated in this study were compared between the two groups.

Results
A second DGC significantly increased the motile sperm recovery rate from poor semen samples. To determine whether a second DGC will increase the recovered sperm from semen, we separated and collected the sperms from normal and poor semen samples by first and second DGCs, and the parameters of sperm from the first and second DGCs were analysed. The results showed that an amount of sperm could still be extracted from normal or poor semen samples by the second DGC. However, the relative total sperm count and the ratio of PR-sperm acquired by the second DGC to the first DGC were much higher from poor semen samples than those from normal semen samples ( Fig. 1A-C). These results indicated that the second DGC effectively separated motile sperm from poor semen samples. Basic characteristics of patients. The present study included a total of 529 IVF cycles, 306 cycles for the DGC/SU group and 223 cycles for DDGC/SU. The patient characteristics and cycle parameters were compared between the two groups as shown in Table 1.The percentage of primary infertility, infertility duration, paternal or maternal age and BMI, ovulation induction protocols, basal hormonal levels, total gonadotropin (Gn) use, duration of Gn use, endometrial thickness, average oocytes retrieved and MII oocytes were comparable in the DGC/ SU and DDGC/SU groups ( Table 1). The DNA fragmentation index (DFI) was significant higher in the DDGC/ SU group than that in the DGC/SU group, while the sperm concentration, the proportion of progressive motility (PR) and normal morphology sperm were lower in the DDGC/SU group (Table 1). In addition, the proportion of male-infertility factors was significantly higher in the DDGC/SU group ( Table 1).

comparison of in vitro embryo developmental parameters between the DGc/SU and DDGc/ SU groups.
The results showed that the rate of normal fertilization and the formation rate of top D3 embryos (grade I and II) from either 2PN or 1PN zygotes and blastocysts from either top or not top D3 embryos (grade III) were comparable in the DGC/SU group and DDGC/SU group (Table 2). However, the 1PN zygote formation rate was significantly lower in DDGC/SU group than that in the DGC/SU group ( Table 2). comparison of clinical outcomes of embryo transfer between DGc/SU and DDGc/SU groups. Given that the clinical outcomes for cleavage stage embryo transfer differ greatly from blastocyst transfer, we subdivided groups of DGC/SU and DDGC/SU into D3 embryo and blastocyte subgroups. The number of D3 embryos or blastocytes per transfer was similar in the DGC/SU and DDGC/SU groups ( Table 3). The pregnancy rate, spontaneous abortion rate, implantation rate, live birth rate, birth weight and duration of www.nature.com/scientificreports www.nature.com/scientificreports/ pregnancy from the transfer of either D3 embryos or blastocytes were comparable in the DGC/SU group and DDGC/SU group (Table 3).

Discussion
Our data showed that sperm preparation for IVF cycles by the strategy of DDGC/SU could significantly improve the sperm recovery rate from poor semen samples without adversely affecting the biological processes, including fertilization and in vitro and in vivo embryo development, suggesting that DDGC/SU could be a simple, effective and safe way to separate sperm from poor semen samples for IVF.
A previous study demonstrated that additional centrifugation time or increased centrifugation force may increase the sperm recovery rate 9 . Consistent with this finding, we found that additional centrifugation time increased the recovered sperm from both normal and poor semen samples. More importantly, additional centrifugation can separate more highly motile sperms from poor semen samples than from normal samples. Poor semen samples characterized by high viscosity, unresolved fibres or clots may result in more drag force to retard the downward movement of motile sperms during DGC. Additional centrifugation provided enough power that allowed the motile sperm thoroughly move through the entrapment. In normal semen, motile sperm can be easily separated by first DGC, and the second DGC resulted in the congregation of sperm with limited motile capacity, even immotile sperm. Therefore, a second DGC is more necessary and effective for the separation of sperm from poor semen samples.
As mentioned above, the efficiency of separating sperm from poor semen samples which are characterized by liquefaction defects, containment of too many unresolvable clots or rare sperm after one DGC, is very low. In  www.nature.com/scientificreports www.nature.com/scientificreports/ some cases, due to a low sperm recovery rate, the enriched sperm by one DGC from poor semen samples will not meet the requirement of insemination by conventional IVF, and ICSI would be the substitute. Although the safety of ICSI is generally accepted, a recent systematic review paper showed that compared with IVF-conceived children, ICSI-conceived children may be at increased risk of autism and intellectual impairment and suggested an uncertainty of the long term safety of ICSI 19 . Furthermore, insemination by ICSI will increase the financial burden of patients. Therefore, poor semen samples processed by DDGC/SU help to reduce the total intracytoplasmic sperm injection (ICSI) cycles.
In the present study, although the DFI of fresh semen samples was not taken into consideration when deciding whether the second centrifugation should be performed, we found that the DFI was significantly higher in the DGGC/SU group than that in the DGC/SU group. Previous studies demonstrated that the level of DFI negatively correlated with semen parameters, including the sperm concentration, sperm motility, and normal sperm morphology [20][21][22] . Consistently, we found that the sperm concentration, percentage of PR sperm and normal sperm morphology were lower in the DDGC/SU group. In addition, the male-infertility factor ratio was significantly higher in the DDGC/SU group. The other characteristics of couples undergoing IVF cycles were comparable in the DGC/SU group and DDGC/SU group, including the age and BMI of males and females, type, duration and diagnosis of infertility, ovulation induction protocols, the total dose and duration of Gn use, endometrial thickness, average oocytes retrieved and MII oocytes. These results demonstrated that "poor" semen in the DDGC/SU group showed poor semen quality.
It has been shown that sperm with a high level of DFI negatively correlated with the outcomes of IVF-ET 18,23 . However, the findings from other studies suggested that DFI was not able to predict the outcomes of IVF [24][25][26] . Although no consistent conclusion was made, it is at least clear that sperm with high-level DFI will not contribute to improving the outcomes of IVF cycles. In the present study, although a higher DFI of semen was present in the DDGC/SU group, the indicators including the normal oocyte fertilization rate, top D3 embryo formation rate, blastocyte formation rate, pregnancy rate, implantation rate, live birth rate, birthweight and duration of pregnancy from either the transfer of D3 embryos or blastocytes were comparable in the DGC/SU group and DDGC/ SU group. It has also been reported that sperm DFI positively correlated with spontaneous abortion [27][28][29] . In the present study, we found that the spontaneous abortion rate following the transfer of either D3 cleavage embryos or blastocytes was comparable between the two groups. A recent study indicated that only DFI over a "threshold" may increase the risk of spontaneous abortion and suggested that each laboratory may have its own threshold of DFI 27 . Therefore, we speculated that although a significantly higher DFI was observed in the DDGC/SU group, the DFI may not reach the threshold to induce spontaneous abortion. These results demonstrated that sperm from poor quality semen samples with higher DFI by DDGC/SU for IVF achieved the same outcomes as sperm from normal semen by DGC/SU, suggesting that DDGC/SU is a safe method for sperm enrichment for IVF.
Previous studies in rodents showed that increased centrifugation force would damage the cell organelles and membranes of sperm [10][11][12] . However, few studies have been conducted to further explore the effect of the sperm suffering excess centrifugation force on the outcomes of IVF in any species. Only one study showed that rat sperm with over centrifugation had no impact on the fertilization rate 12 . In line with that, we also found that double centrifugation did not decrease the fertilization rate, nor the other indicators mentioned above. It is known that DNA, located in the head of sperm, is highly compacted, and the most important role that sperm plays in fertilization is to transfer its DNA into oocytes. In the present study, we speculated that increased centrifugation force was not likely to damage sperm DNA, but to cause limited membrane or organelle damage that was far from affecting sperm motile capacity.
The formation of the 1PN zygote may be associated with several causes, including the fusion of male and female pronucleus, the asynchronous disappearance of male and female pronucleus, and only male or female pronucleus formation 30,31 . Theoretically, the first 2 causes will not impair further embryo development while the latter 2 will. To our surprise, we found that poor semen processed by DDGC/SU significantly decreased the ratio of 1PN zygote formation without affecting the ratio of top D3 embryo formation derived from 1PN zygotes, when compared with DGC/SU for normal semen, suggesting that poor semen processed by DDGC/SU synchronously decreased all causes-resulted the formation of 1PN.
In summary, our study verified that DDGC could effectively improve motile sperm recovery from poor semen, and showed that sperm for IVF from poor quality semen samples processed by DDGC/SU were able to achieve the same outcomes as sperm from normal semen by DGC/SU, suggesting that DDGC/SU is an effective and safe method of the treatment for poor semen samples in IVF, that is, poor semen samples can be processed by DDGC/SU for IVF in the clinic.

Materials and Methods
Study subjects. The data generated from a total of 529 IVF cycles (from June 2017 to June 2018) and these IVF cycle-contributed 622 transfer cycles (from June 2017 to December 2018) in Changzhou Maternal and Child Health Care Hospital, were collected and analszed in this retrospective study. Subject to semen preparation, data from IVF cycles or ET cycles were divided into two groups: the DGC/SU group and the DDGC/SU group. All of the included patients read and signed the informed consent form. This retrospective study was approved by the Ethics Committee of Changzhou Maternal and Child Health Care Hospital and Nanjing Medical University. All the treatments in the present study were performed strictly in accordance with the Declaration of Helsinki for Medical Research.
Semen preparation. Patients with 2-5 days of abstinence ejaculated semen into a sterilized cup by masturbation. After liquefaction, 10 µl of semen sample was drawn on the slide and covered with a cover glass to roughly estimate sperm concentration and sperm motile parameters, including the percentage of progressive motility (PR) sperm, non-PR (NP) and immobile (IM) sperm, under a microscope with a magnification of 400X. (2020) 10:2286 | https://doi.org/10.1038/s41598-020-59347-y www.nature.com/scientificreports www.nature.com/scientificreports/ The sperm concentration (10 6 /ml) was roughly estimated as sperm counts in a 400X visual field. The remaining sample was placed on a density gradient column consisting of 1.5 ml of lower layer and 1.5 ml of upper layer (Irvine, USA) and centrifuged at 462 g for 15 min. For DGC/SU, the sperm pellet was re-suspended in 3 ml of IVF plus medium (Vitrolife, Sweden) and washed by centrifugation at 370 g for 9 min. After that, the sperm pellet was re-suspended in 0.5 ml of IVF medium. Ten microliters of medium was used to roughly estimate PR sperm counts. Then, 0.5 ml of IVF medium was dropped gently and slowly on the surface of the sperm suspension along the inner tube wall, and the tube was placed at a vertical angle in an incubator at 37 °C and 5% CO 2 for approximately 1.5 h before insemination for the purpose of swim up. For DDGC/SU, after the transfer of the sperm pellet obtained from the first DGC, the density gradient column was centrifuged immediately for another round. The sperm pellet from the second DGC was then mixed with the sperm pellet from the first DGC. The remaining procedures were the same as the abovementioned DGC/SU. A work flow of DDGC/SU is described in Fig. 2. For insemination, a micropipettor was used to transfer the sperm into the culture drop of the oocyte.
inclusive criteria of semen for DDGc/SU. In this study, semen were processed by DDGC/SU if (a) semen showed liquefaction defects, or contained too many unresolvable clots/fibres, or contained rare but high motile sperm, and (b) the sperm pellet after the first DGC was invisible or almost invisible.
Computer-aided semen analysis system (CASA) analysis. Ten microliters of semen was added to the chamber of slides, and after a warm-up (10 min), the slide was analysed by CASA. The sperm concentration and motility-related parameters were recorded. The results of semen quality parameters presented in Table 1 and results 1, including the sperm concentration, total sperm count and ratio of PR sperm, were determined by CASA analysis.
iVf procedures. More than half of the patients received ovarian stimulation with GnRH analogues, and the rest were treated without GnRH analogues for controlled ovarian hyperstimulation. Insemination was performed in IVF plus medium (Vitrolife, Sweden) using conventional IVF. Approximately 16-18 h after insemination, oocytes with 1 or 2 visible pronuclei were further cultured in G1 plus medium (Vitrolife, Sweden). On day 3 of culture, embryos were scored as grade I, II, III or VI based on the number, size, and shape of blastomeres and their degree of fragmentation, as previously described 32 . Grade VI embryos were discarded. D3 embryos were subjected to transfer, frozen by vitrification technology or expanded in G2-plus culture medium (Vitrolife, Sweden). Blastocysts from day 5 or 6 were scored using the system of Gardner. The usable blastocysts were frozen by vitrification technology or transferred. Statistical analysis. All analyses were performed by using GraphPad prism software (version 5). The chi-square test was used to compare the data of the constituent ratio. If continuous variables were normally distributed, Student's t-test was used. If not, the Mann-Whitney U test was used. P < 0.05 was considered statistically significant. Figure 2. Work flow of DDGC. A total of 6 consecutive steps were involved in the entire process. Briefly, semen was placed on the density gradient column in the tube for the first DGC. After the first DGC, sperm precipitation was transferred into IVF medium. The semen in the tube on the column was subject to the second DGC. The sperm precipitation from the second DGC was also transferred to the abovementioned IVF medium. The sperm were then washed by centrifugation and re-suspended in 0.5 ml of IVF medium. Finally, sperm underwent swim-up procedure.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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The Importance of Crop Rotations for Wild Oat (Avena sterilis L.) Resistance to Herbicides in Cukurova Region, Turkey

Received: 26 October 2020 Revised: 24 November 2020 Accepted: 28 November 2020 Available online: 04 December 2020 DOI: 10.26655/JRWEEDSCI.2021.1.10 This study was conducted between 2013 and 2015 to determine the changes in the populations of wild oat (Avena sterilis L.) coverages, resistance and weed coverages based on the crop rotation applied at fields where wheat was being grown in Osmaniye Province and to reveal the relationship between the crop rotations and the resistance developed in wild oat by Clodinafop-propargyl with the Acetyl-CoA Carboxylase (ACCase) mode of action, Mesosulfuron-methyl + Iodosulfuron-methyl sodium and Pyroxsulam of the Acetolactate Synthetase (ALS) effect mechanism. By determining crop rotation systems for May and August in 103 fields selected in the year 2013, the coverage changes in the populations of wild oat and weeds and the resistance in wild oat poulations were determined. As a result, 48 different crop rotation systems were determined, wild oat resistance disappeared in the wheat + sunflower + sunflower rotation which was one of the most frequently preferred rotation, while the resistance index (RI) of wild oat against the ACCase inhibitor fell (RI<2.00) in the two-crop rotations involving maize as wheat/maize + maize + maize, wheat/maize + wheat/maize + wheat, wheat + wheat/maize + wheat and wheat/maize + wheat/maize + maize. In the three-crop rotation systems, resistance against ALS decreased in wheat/peanut + wheat/sesame + wheat, and resistance against ACCase decreased in wheat/maize + maize/peanut + peanut. It was revealed that the producers did not prefer four crop rotation much, and fallow did not change resistance indices. The wild oat resistant populations and coverages in the Osmaniye Province were determined for the first time, and the interactions of crop rotations are presented.

Introduction
Whatever weed management or integrated management system is used, human activities and survival interactions with other species of the production system have always led to the emergence of new weed species in agriculture and the environment. In addition to alternative methods used for weed management, chemical herbicides are the most economical ones (Bridges, 1994). Yield losses in production caused by weeds, a worldwide problem, amount to 13.2% or 75.6 billion dollars per year (Oerke et al., 1994). In the United States alone, weeds account for 12% of the annual loss of crops and about 36 billion dollars of monetary losses. Furthermore, four billion dollars are spent on herbicides every year to control weeds (Pimentel et al., 1999). Cereals, main foods among cultivated plants, have great importance in Turkey as in everywhere around the world. The increase in cereal fields in Turkey has continued to the present day, and it has reached over 120 million acres in total. Some of the main limiting factors in production, like with other cultivated plants, are weeds (Wilson et al., 1990). Wild oat species from the Poaceae family, found in cereal fields in almost all countries around the world, are the main major harmful weed species for wheat (Kadıoğlu et al., 1990;Torner et al., 1991). In order to control wild oat species (Avena spp.) worldwide, the use of post-emergence ACCase and ALS inhibitor herbicides is increasing (Beckie et al., 2002;Owen and Powles, 2009;Torun, 2017). The most widely used herbicides for chemically controlling cereals are the ACCase (Acetyl-CoA carboxylase) and ALS (Acetohydroxy acid synthase) mechanisms, and excessive employment of these chemicals for wheat has shortened their lifespan in Turkey. One of the best examples is the problem of weed resistance against some herbicides in the Cukurova region (Yücel, 2004;Avcı, 2009;Ayata, 2014;Gürbüz, 2016;Torun and Uygur, 2019a). The resistance of wild oats (Avena sterilis L.) and foxtail (Alopecurus myosuroides L.) started locally in some wheat fields in the Cukurova region at the end of 1990s and has been observed to have spread during the 2000s. In the following period, a countrywide resistance of wild oats emerged in Turkey as it has been experienced all around the world (Uludağ, 2003;Türkseven, 2011;Torun and Uygur, 2019a).
The first aim of this study is to determine wild oat population coverages and resistance at fields in the Osmaniye Province between 2013 and 2015 against ACCase and ALS inhibitor herbicides.
The other purpose of the study is to reveal weed coverages and crop rotation systems. Moreover, this study investigated that all the interaction changes which crop rotation system affected the weed population and the problematic wild oat population, mostly in determined fields and find out the selection of three year crop rotation alternatives to decrease coverages and resistance

Determination of wild oat population resistance
The studies were carried out between 2013 and 2015 in the greenhouses of the Department of Plant Protection at the Cukurova University. The main materials of this resistant study were mature wild oats seeds from sampling points in the Cukurova region of the Osmaniye province (Table 2). Tests were performed with rechargeable backpack sprayer working at a constant pressure of 3 atm (Matabi), and bark papers were used during greenhouse experiments. The three year temperature averages were above 25°C and relative humidity 65%. And also resistance tests were set during sunny days in October.
The study was performed using the random blocks test design, with two replicates and four replications. The soil to produce the material used in the study was obtained at places where no herbicides had been used previously, and the soil material put in the trays had a 1:1:1 ratio of sand, fertilizer and soil. Every pail used was 5x5 cm. The prepared soil mixture was filled with equal amounts of seeds and then placed in the greenhouse. The seeds of the wild oats collected between 2013 and 2015 were inspected by spreading about 5 seeds per tray pit and watering the trays for plant germination.
After one month, 3 of the wild oats, which were in their 2-4 leaf period were left in each of the tray pits.
Then, herbicides (200 ml ha -1 Clodinafop-propargyl-ACCase inhibitor, 300 g ha -1 Mesosulfuron-methyl + Iodosulfuron-methyl sodium and 250 g ha -1 Pyroxsulam-ALS inhibitors) were applied to 10 m² areas by randomly applying the rechargeable backpack sprayer. The dry biomass of the weeds was measured prior to application of the herbicides and on day 1 and day 28 after its application. The weeds were harvested from the soil surface at the end of the 28th day when the effect of the herbicides was fully detectable. The plant material was then placed in paper bags, dried for 24 hours at 105°C, and then, weighed for dry biomass (Hitchcock, 1931). Six different doses of herbicides (N/4, N/2, N: Recommended dose, 2N, 4N, 8N, 0: Control) were applied to the weeds susceptible to resistance. During the greenhouse experiments, the populations of wild oats were dispensed with clodinafop, mesosulfuron + ıodosulfuron and pyroxsulam. The formula for the four-parameter dose-response curve was as follows: (1) Formula C: lower limit, D: upper limit, b: slope and ED50: the dosage causing 50% reduction (1) using the Curve Expert Program (Seefeldt et al. 1994;Seefeldt et al. 1995). The recommended dose was compared to the untreated control plot by applying the herbicide at a rate of 50% of the population and at a higher value. Resistance index (RI) was calculated as the ED50 value of the resistant population divided by the ED50 value of the susceptible population. Resistance levels were classified as no resistance (RI < 2.00) and resistance (RI ≥ 2.00) (Beckie and Tardif, 2012;Moss et al. 2007;Moss et al. 2019).

Results and Discussion
As combinations of the rotation systems in the region, a total of 48 different crop rotation were identified in the Osmaniye Province at 103 sampling points between 2013 and 2015. Thereof, the most prevalent rotation systems were wheat + wheat + wheat by 14.56%, wheat/corn + corn + corn by 8.74% and then wheat/corn + wheat/corn + wheat by 5.83%. This clearly showed that there were multiple crop systems in the Osmaniye Province, but sowing different crops was not preferred much (Table 3). From the viewpoint of different crops, it may be determined that the sowing rates were very low for sunflower which can be alternated with wheat, as well as peanuts, soybean and sesame as alternative crops to corn. However, within the crop rotation systems, the wheat + sunflower + sunflower combination was used at 4.85%, and wheat/corn + corn/peanut + peanut rotation was approximately 3.00%. Wheat and corn crops were determined to be preferred over almost all seasons, although the yield of different crops was the subject of the question, and the percentages of cultivation of other systems were found to be between 0.97% and 1.94% (Table 3).

Crop rotation effects towards weed and wild oat coverages and the resistance index One crop rotation
Fifteen fields used wheat as the only cultivated crop in one crop rotation. While looking at fields with monoculture agriculture, only one field combination was observed within 15 fields, and its sowing percentage was 14.56% in total. Especially in wheat + wheat + wheat combinations, an increase of weed coverage and wild oat coverage was clearly detectable (Torun and Uygur, 2019b  Average resistance indexes were given for sampling field numbers. *Pyroxsulam was not tested in 2013.

Two crop rotation
The total number of field combinations obtained with two crop rotation was 17 out of the 49 fields, and their total usage rate was 47.58%. While looking at the sampling fields, it could be observed that the producers gave importance to especially wheat and corn crops. The weed coverage rose at most from 19.25% in 2013 to 47.50% in 2015 with an increase of 146.75% (2.47 times) the for wheat/peanut + wheat + wheat combinations, and it was observed that peanut did not decrease the weed coverage. The combination of wheat/corn + wheat + corn was observed with a 93.75% decrease in weed coverage in the last year in comparison to the first year. Because of hoeing and sowing as a first crop, corn caused coverage changes in the fields (Table 5). As in other agricultural areas of Turkey, a failure of crop rotation systems with some herbicides has caused an increase of the population frequency and resistance formation of significant weed species in wheat cultivation in the Cukurova region (Yücel, 2004;Avcı, 2009;Ayata, 2014;Gürbüz, 2016;Torun and Uygur, 2019a).  (Harker et al. 2009).
The wheat + sunflower + sunflower, wheat/corn + wheat/corn + corn, wheat/corn + corn + corn, wheat + corn + corn and wheat/corn + wheat + corn combinations reduced the wild oat coverage most. Corn was determined during all sowing seasons in two crop system. The wheat/corn + wheat/corn + wheat and wheat + corn + wheat combinations with lower reduction coverage rates in the population followed these ( winter wheat + fallow and spring wheat + winter wheat + corn + sunflower combinations, whereas they also determined that weed germination was 6 times higher with a winter wheat + grey millet rotation compared to these rotations. They revealed at least 4 crops for rotations in weed management. A study in Iran examined the effects of rice sown with different crop rotations with rice and rice + soybean + rice combinations. Rice + soybean + rice was determined to reduce weed density by 62.50% and by 80.00% for the weed biomass, and crop rotation increased the rice yield (Filizadeh et al. 2007).
The wheat + sunflower + sunflower system's wild oat RI was found to be 4.00 with the ACCase herbicide, and the 3.11 ALS Pyroxsulam herbicide disappeared during the last year. For wheat/corn + wheat/corn + corn rotation, the ACCase RI was 3.67 in 2013, but during the last year, no resistance was observed in the fields. In the wheat/corn + wheat + corn system, resistance was not observed in 2015, whereas the resistance with wheat/corn + corn + corn against Mesosulfuron + Iodosulfuron was found to be 5.15 in the last year (Table 5). Gürbüz (2016) identified ALS inhibitor resistance in Adana against wild oat and wild mustard (Sinapis arvensis L.) populations. They found that 90 wild oats and 22 wild mustard populations were resistant to Pyroxsulam, while 37 wild oat and 26 wild mustard populations were detected to be resistant to Mesosulfuron + Iodosulfuron. In 2015, the resistance of wheat/peanut + wheat + wheat against Clodinafop and the resistance of the wheat + wheat/corn + wheat, wheat/corn + wheat/corn + wheat and wheat/corn + corn + wheat combinations against Mesosulfuron + Iodosulfuron were determined (Table 5). In Canada, Beckie and Kirkland (2003) reported that different crops had decelerated resistance, and they revealed that limited crop sowing over four years strengthened the population of susceptible individuals.
The wheat + fallow + wheat and wheat + fallow + fallow combinations showed a decrease in the coverage of weeds and wild oat during the last year in comparison to the first year. However, there was no benefit of laying the fields fallow, because resistance against the ACCase or ALS herbicides was determined in these systems, or it increased again (Table 5). A sampling of 62 wild oat populations was picked in Canada, and the ACCase inhibitor Imazamethabenz and the ALS inhibitor Triallate were applied to test the resistance levels for confirmation. Because of using the same herbicides in the fields with no crop rotation, a resistance clearly occurred (Beckie et al., 2004).

Three crop rotation
The highest number of fields used a combination of 27 crops in three crop systems. At 103 sampling points, 34.95% of the total sowing percentage was seen in 36 fields. It was observed that even in the case of three different crops, resistance was constant or increased with some crop rotations, despite a decrease in the coverage of weeds and wild oats (Torun and Uygur, 2019b). In the Cukurova region, Yücel (2004) observed different levels of resistance to ACCase inhibitors and tested wild oat populations with the ALS inhibitor Mesosulfuron whose RI was found to be 11.34. In another study on wild oat populations from the Eastern Mediterranean Region of Turkey, it was revealed that Fenoxaprop usage was intense, and resistance was determined in wild oat populations (Uludağ, 2003). The weed coverage of the wheat/soybean + wheat/corn + wheat combinations showed an increase of 2.50% to 26.00%, totalling 940% (10.4-fold), the whereas wheat/corn + peanut/corn + wheat combinations contrived a decrease of 96.25%, from 80.00% to 3.00%, during the last year ( Table 6).
The highest increase in the wild oat coverage was observed in the wheat/peanut + wheat/corn + wheat combinations with 266.67% (3.67-fold), increasing from 7.50% to 27.50%. The wheat/soybean + corn/soybean + corn, wheat/peanut + corn/peanut + peanut and wheat/corn + peanut/corn + wheat combinations caused a 100.00% decrease in wild oat coverage (Table 6). Preston and Powles (2002) deduced ryegrass (Lolium rigidum L.) population changes with the ALS inhibitors Sulfometuron-methyl and Imazapyr in organic farming in Australia. They exposed collected susceptible populations to herbicides, and so, they detected that susceptible populations could easily resist over shorter times. Owen and Powles (2009)   Additionally, the Clodinafop RI in the wheat/sesame + wheat/soybean + wheat combinations ranged from 4.67 to 1.67, and the RI for wheat/corn + corn/peanut + peanut decreased from 2.44 to 1.35. In many European countries, slender meadow foxtail (Alopecurus myosuroidesHuds.) was found to be resistant to ACCase and ALS inhibitors. To prevent resistance, one should (1) reduce the use of ACCase and ALS inhibitor herbicides that are employed due to a development of herbicide resistance, (2) use different mechanisms of action by identifying different weed management strategies and mix herbicides at different ratios against resistance formation and (3) employ cultural measures such as ploughing, crop rotation and delayed tillage (Moss et al. 2007).
The Mesosulfuron + Iodosulfuron effect towards the wheat/corn + wheat/peanut + corn RI was constant, but resistance increased at the end of three years for wheat + sunflower + barley to 2.44, for wheat/peanut + wheat + barley to 2.43, for wheat/corn + wheat/corn + peanut to 3.33, for wheat/sesame + wheat/soybean + wheat to 2.47 and for wheat/soybean + corn + corn to 2.37 (Table 6). Based on rotation systems with three different crops, Koocheki et al. (2008) conducted some comparative research in Iran and determined that seed density was 26.00% higher in fields where only wheat was grown. While the density of grass weeds in wheat fields was 90.00%, this density decreased to 43.00% in the wheat + sugar beet rotations. Besides, the seed density was also found to be lower in the wheat + corn and wheat + sugar beet rotations. In Australia, Martin et al.  (Table 6). Cardina et al. (2002) looked at only corn, corn + soybean and corn + wheat + dry grass rotations in three different rotation systems at different locations. They found 40.00% more seed bank reserves in fields of only corn in comparison to corn + wheat + dry grass rotations.
In crop rotation fields, the number of seed bank reserves and seeds in soil were found to be the least in America.

Four crop rotation
Four different crops were found to be sown at only 3 fields along with all sampling sites. It was observed that there were 3 different combinations sown in total, amounting to 2.91%. Four different crops did not reduce the resistance against any ACCase or ALS inhibitor, while also not reducing the weeds and wild oat coverage ( Table 7). Cultivation of the same crops in the same fields, as in a study by Tardif and Powles (1993), has shown that resistance to ACCase inhibitors used against wild oat populations may be seen in less than three years. Due to steady applications of tillage, irrigation or fertilizers in the fields, the wild oat density in the wheat fields sustained their detrimental effect and became even more harmful except for fallow periods. Average resistance indexes were given for sampling field numbers. *Pyroxsulam was not tested in 2013. **Not enough population. F: Fallow counted as a crop.
The RI of wheat/corn + soybean/peanut + corn was constant, but a Mesosulfuron + Iodosulfuron resistance was determined for the wheat/corn + fallow/sesame + wheat and wheat/corn + fallow/peanut + wheat combinations. In the last year, the RI against Clodinafop was found to be 8.93 for wheat/corn + fallow/sesame + wheat (Table 7). In Romania, Pop et al. (2009) observed that 305-365 weeds/m 2 were found in corn + wheat rotation areas, whereas 213-236 weeds/m 2 were observed for soybean + corn + wheat + sunflower combinations. Liebman and Dyck (1993) addressed crop rotation and cultivation of different crops in terms of sustainable agriculture and noted that application of crop rotation acts like an automated control system for wild oats, and the density of harmful wild oats decreases.

The effects of herbicide resistance on wild oat populations during transition to polyculture crop rotations from monoculture crop rotations
Continuously sowing the same crops increases the frequency of weeds and wild oats, and only an increase in the number of different crops may cause a reduction of these frequencies. The most important reason is that application of contention mechanisms against weeds in cultivated crops is limited. For example, while chemical control is applicable exclusively for wheat fields, hoeing is important in reducing the density of existing weed species in crops such as corn, peanut and sunflower. In monocultural rotation, using the same crops in the same fields, a sampling between 2013 and 2015 showed an increase in the weed and wild oat frequency and weed resistance, while variable cropping with two, three and four crops decreased the resistance (Liebman and Dyck, 1993;Anderson et al. 2007;Filizadeh et al. 2007;Owen and Powles, 2009;Pop et al. 2009).
The most preferred crop rotation, wheat only over three years, increased the coverage of weeds and wild oats, as well as increasing the resistance against herbicides, especially ACCase and ALS inhibitors, eventually leading to removal of wheat sown in these fields. For wheat + wheat + wheat, resistance against only Clodinafop was detected every year. However, addition of sunflower, an alternative to wheat, and employing wheat + sunflower + sunflower combinations resulted in reduction of wild oat coverages and even a break in resistant populations. Because of changes in tillage, irrigation or fertilizer use in wheat fields, the possibility of wild oat population resistance decreases (Table 8). One study showed that the same crop sown in a field increases the resistance of wild oat populations against ACCase inhibitors in less than three years (Tardif and Powles, 1993). For the wheat + sunflower + sunflower combinations, although the weed coverages increased, the wild oat population decreased in the last year due to cultivation of sunflowers. The resistance against the herbicide Clodinafop disappeared (Liebman and Dyck, 1993;Blackshaw et al. 2001;Cardina et al. 2002;Anderson et al. 2007;Owen and Powles, 2009;Pop et al. 2009;Ahmad-Hamdani et al. 2013;Torun and Uygur, 2019a) (Table 8).
The one crop system of wheat instead of corn and peanut caused a consistency of the existing resistance or created new resistance. The coverages decreased in wheat/corn + corn + corn rotation systems, which were two-crop rotations. However, resistance against ALS Mesosulfuron + Iodosulfuron was detected. This was because the resistance was triggered by the fact that the current herbicides used with corn employ the same mechanism of action. In a similar study, Beckie and Kirkland (2003) concluded that different crop rotations discouraged resistance in certain wild oat grass populations, which are resistant to herbicides, and the resistant individuals became much stronger among the resistant and sensitive populations as a result of cultivated crops limited to a four-year application period. Because of hoeing in combinations of corn and peanut in wheat/corn + wheat/corn + wheat, wheat/corn + wheat/corn + corn and wheat/corn + corn/peanut + peanut with three crop rotations and because wheat, which is the main host of wild oat, cannot be grown in May, the coverage of the population of weeds and wild oats decreased in the last year (Blackshaw et al. 2001;Anderson et al. 2007;Harker et al. 2009;Pop et al. 2009). The resistance to the ACCase inhibitor in the wheat/corn + wheat/corn + wheat combination was persistent only in the last year. It was also determined that wheat/peanut + wheat/sesame + wheat, which is a three-crop rotation with peanut and sesame cultivated after wheat, did not affect the coverages in the rotations. In 2015, resistance to Clodinafop and Mesosulfuron + Iodosulfuron was detected in wheat. Wheat/corn + corn/peanut + peanut showed the reason for the decrease in wild oat coverage as the fact that introduction of different crops in the crop rotation does not constitute resistance (Torun and Uygur, 2019b). In wheat/peanut + wheat/sesame + wheat, the coverage of weeds and wild oats decreased, but resistance against Clodinafop was recorded (Table 8).

Conclusion
In determining crop rotation systems in fields sowing wheat has continuously developed the coverages and resistance in one and two crop rotation between 2013-2015 in Osmaniye province. It shows that the use of ACCase and ALS inhibitors herbicides should be highly used and preferred in wheat fields. Because of limiting licensed same effects mechanisms against wild oats in wheat is critical level where resistance has been created and rised in Osmaniye Province and ensure its sustainability in a long time is necessary. In some rotation systems have been observed that the number of resistance indices may decrease over time in three years. Of course managing weeds and wild oat populations with the diversification of field crops have decreased the coverages and frequencies on a different date of irrigation, fertilization and tillage times. Monoculture one crop rotation includes wheat have developed the resistance of wild oats and is also insufficient to decrease to resistance levels. Polyculture crop rotations have decreased the resistance indices. Moreover, correct and long crop rotation systems for fields must apply and need to be planned. Instead of cultivating the same crop in the fields in the short term, planned crop rotation systems with different field crops are healthy, reliable and correct in the long term with its rotation should be contributed. Crop rotation and agricultural ecosystem should be together for sustainable agriculture.

Conflicts of Interest
The authors have declared no conflicts of interest.


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Roles of arbuscular mycorrhizal fungi on acclimatization of clones of Coffea arabica L. produced by somatic embryogenesis

The production of Coffea arabica L. clone seedlings through the somatic embryogenesis technique is one of the alternatives of greatest demand for coffee producers. However, clones can face difficulties related to acclimatization carried out under greenhouse conditions, which can increase the production costs. A tested alternative with promising results is inoculation with arbuscular mycorrhizal fungi (AMF) during the seedling acclimatization process. Thus, this study aimed to promote the improvement of the seedling production process through somatic embryogenesis associated with inoculation with AMF. For the production of the clones, seedlings were used in the phase when they presented four pairs of leaves (Clone 1 “Fruiting red Catucaí” and Clone 2 “Acauã”), using “somatic embryogenesis” bioreactors, followed by inoculation with Rhizophagus clarus, Gigaspora margarita and Acaulospora mellea, a mixture of R. clarus and G. margarita and a mixture of R. clarus, G. margarita and A. mellea. After six months, agronomic parameters, leaf nutrient contents and root mycorrhizal colonization were evaluated. The studied clones behaved differently when inoculated with AMFs. Clone 1 showed the best development in the greenhouse, which was determined by the agronomic parameters. Thus, Clone 1 is indicated for the production of vigorous seedlings when associated with inoculation with AMFs.

INTRODUCTION
Coffee is a crop of significant importance for the Brazilian agribusiness. It promotes economic and social development, generating jobs in the coffee producing regions (Embrapa, 2006). The State of Minas Gerais stands out as the largest Brazilian producer of Coffea arabica L., contributing to more than half of the total national production (Conab, 2020).
Lately, new methods to increase coffee productivity and quality have been pursued by Brazilian coffee growers and researchers. The production of clones of hybrids of C. arabica L. is one of the most promising alternatives (Etienne et al., 2002). Besides the increase of over 30% in productivity when compared with traditional cultivars, it is also possible to add features of interest more quickly than traditional methods related to the obtention of plant varieties (Bertrand et al., 2005).
As an alternative to the multiplication of F1 hybrids, somatic embryogenesis stands out among the methods of vegetative propagation. Small amounts of source material (leaves) are used for the production of high numbers of clones (Rezende et al., 2012). However, clones of C. arabica L. produced via somatic embryogenesis can present some post-laboratory complications, such as the high cost for the production of clones and the difficulties considering their acclimatization (Etienne et al., 2002;Carvalho et al., 2013). Thus, the use of arbuscular mycorrhizal fungi (AMF) in micro-propagated clones of C. arabica L. can be a promising technology.
Arbuscular mycorrhizal fungi (AMF), belonging to the phylum Glomeromycota, establish symbiotic mutualistic associations with the roots of most vascular plants (Siqueira et al., 2010). These fungi colonize greater volume of soil due to their mycelium network, increasing the volume of exploited soil and absorption of water and nutrients, especially phosphorus (Maia;Silveira;Cavalcante, 2006;Kahiluoto;Ketoja;Vestberg, 2012;Posada et al., 2018;Rojas et al., 2019). Furthermore, it provides greater resistance to adverse environmental conditions by increasing the post-planting survival and the initial development of coffee trees in the field (Silveira et al., 2003;Cardoso;Barreto, 2006;Fonseca et al., 2019). The symbiosis also increases the absorption of nutrients such as P, Ca, Mg, S, and Cu and protects against toxicity of Mn, and Zn (Saggin-Junior et al., 1995;Saggin-Junior;Siqueira, 1995), promoting greater increments on dry mass and nutrient accumulation (Prates Junior et al., 2019).
The increased efficiency of the production process of clones via somatic embryogenesis using AMF can reduce costs in the production of vigorous coffee seedlings. The use of AMF in coffee seedlings is still little studied, as demonstrated by Cogo et al. (2017) in their systematic review. Therefore, this study aimed to evaluate the contribution of AMF on the acclimation of two clones reproduced by somatic embryogenesis.

MATERIAL AND METHODS
The experiment was conducted under greenhouse conditions at the experimental nursery of Procafé Foundation, located in Varginha, state of Minas Gerais, Brazil (geographical coordinates 21°34'00"S and 45°24'22" W, altitude = 1,000 m).
The treatments were composed of four repetitions and six clones per each experimental unit. The studied clones were chosen based on their resistance to rust (Hemileia vastatrix), good quality of beverage and high productivity. The genotype Red fruiting Catucaí (Clone 1) originates from the crossing between Icatu and Catuaí and Acauã (Clone 2) originates from the crossing between 'New World 388-17' and 'Sarchimor IAC 1668'.
For the multiplication of AMF, a substrate composed of soil (Dystrophic Red Latosol) and sand at a ratio of 1:1 (v:v) was used. The substrate was sterilized by autoclaving at 120 °C for 60 minutes and this process was repeated by completing 24 hours. After 20 days, 3-L pots were filled with substrate and seeded with Brachiaria decumbens, which was inoculated separately with spores of R. clarus, G. margarita and A. mellea. After cultivation, the spore density of each species was quantified separately, using 50 mL of sample, followed by wet extraction, according to (Gerdemann;Nicolson, 1963).

Clones and substrate production and fertilization
The plants used in this study were produced by somatic embryogenesis, according to the protocol described by (Teixeira, 2018). Leaves from the matrix plants that were disinfected were cut into small segments and placed in Petri dishes with culture medium for the induction of embryogenic calluses. The embryogenic calluses were multiplied in a liquid medium under constant agitation, followed by induction to regenerate somatic embryos. The somatic embryos were transferred to temporary immersion bioreactors and maintained in this condition until the cotyledon stage. The embryos were grown in pots with culture medium for seedling development. When the clones presented about five centimeters in height and four pairs of leaves, they were transferred to polyethylene tubes of 290 cm 3 with an inert substrate composed of thin coconut fiber, and stored under greenhouse conditions to be acclimatized.
The substrate fertilization for the treatments with and without (control) inoculation of AMF was carried out by adding 8.00 g dm -3 of 14-14-14 fertilizer (N-P-K) of slow-release Osmocote ® and 5.00 g dm -3 of 09-45-11 fertilizer (N-P-K) of Raizal 400 ® to the substrate. At the end of the test, the substrates of the treatments with and without inoculation with AMF were analyzed for their nutrient contents, presenting the following results without fertilization: 1.4 mg dm -3 P, 3.3 mg dm -3 K, 3.0 cmol c dm -3 Ca and 0.6 cmol c dm -3 Mg. After fertilization, the substrate presented the following values: 27.3 g kg -1 N, 2.3 g kg -1 P, 14.9 g kg -1 Ca and 3.0 g kg -1 Mg.

Inoculation of seedlings
During the process of pricking, 336 seedlings were transferred from in vitro conduction to tubes of 290 cm 3 . At the time of transplanting, the clones were inoculated with 50 mL of AMFs using soil inoculum containing 600 spores, hyphae and colonized roots composed of R. clarus, G. margarita, A. mellea, and mixtures of A. clarus + G. margarita and R. Clarus + G. margarita + A. mellea. The same proportions of spores were applied in the mixtures.

Experiment conduction
The tubes with the seedlings were placed in a greenhouse equipped with nebulization irrigation system, within 12 hours, on for 20 seconds with a 20 minutes watering shift, and water blade of 0.8mm, daily. The seedlings remained in the greenhouse for 135 days. After this period, they were transferred to a nursery for acclimatization during 180 days.
The acclimatization of the seedlings was held in nursery equipped with protection on the top and on the sides with 50 % shading screen (Sombrite ® ). An automatic intermittent sprinkling irrigation period of 4 hours, connected by 5 minutes with irrigation shift of 5 hours and daily water blade of 6 mm was used.
After six months, the experiment was evaluated with ClorofiLoG ® model CFL 1030 chlorophyll-meter, expressed as Falker Chlorophyll Index (FCI). The leaf areas of the seedlings were also determined (Huerta, 1962). The material was cut and separated into roots and shoots.
The roots were washed in running water for removal of substrates, and fragments of 1 to 2 cm were stored in 70% alcohol solution for later determination of the percentage of colonized roots (Giovannetti;Mosse, 1980). The remaining roots as well as the whole aerial part were dried until constant weight in a forced air circulation oven at 65 °C for the determination of root dry mass (RDM), shoot dry mass (SDM) and total dry mass (TDM).
After determining APDM, shoots were milled in mill type Willey with 40 mesh sieve and digested in sulfuric acid solution for the determination of N (nitrogen) by micro Kjeldahl method (distillation) and nitro-perchloric solution 2:1 (v:v). The contents of of phosphorus (P) and calcium (Ca) were then determined by colorimetry and atomic absorption spectrophotometry, respectively (Malavolta;Vitti;Oliveira, 1997). The accumulation of nutrients was calculated by multiplying the concentration of nutrients in the aerial part by aerial part dry mass.

Statistical analyzes
The data were analyzed for normality (Lillierfors test) and homogeneity of variances (Cochran and Barttlet test). The percentages of colonized roots and leaf area were transformed by using ln (x + 2) when a normal distribution was not followed or when the value was inferior to 30%. The variables were submitted to analysis of variance using the F test at 5% significance level and the averages of the treatments where compared by the Scott and Knott test at 5% significance level. Correlation analysis was determined between the variables at 5% significance level using the Pearson test. All statistical analyses were performed on the statistical analysis system Sisvar (Ferreira, 2011) andSAS program (SAS, 1999), version 9.1.

RESULTS AND DISCUSSION
The height of the aerial part in Clone 1 inoculated with AMF showed higher values when compared with non-inoculated plants (20.4 and 41.7 %, respectively). No significant differences were observed between the treatments in Clone 2. The inoculations of AMF in F1, F2, F3 and F5 in Clone 1 were higher when compared with Clone 2. The stem diameter presented the same trend of the values of height for Clone 1. The seedlings of this clone inoculated with AMF in F1, F2, F3, F4 and F5 showed increases in stem diameter, ranging from 18.6 to 39.5% when compared with the control treatment (Table 1). As for the inoculations of AMF in F1, F2 and F3 in Clone 1, stem diameter values were higher than Clone 2. The leaf area of Clone 2 with and without inoculation with AMF did not differ among the treatments while Clone 1 presented higher leaf area values for the inoculation treatments when compared with the control treatment. This increase ranged from 55.5 to 103.3%. When comparing the effects of the studied clones, Clone 1 inoculated with AMF in F1, F2, F3, F4 and F5 showed increases when compared with Clone 2.
The present study corroborates with the proposed in the literature for the initial growth of seedlings of coffee, indicative that the AMF species used as inoculants promoted a positive response . Some authors observed that cultivars of C. arabica L. inoculated with species and/or isolates of AMF feature significant increase for the characteristics of height and stem diameter (Bhattacharya;Bagyaraj, 2002;Konrad et al., 2014).
It is clear from these results that there is a positive response to the inoculation of seedlings with AMF, especially for height, stem diameter and leaf area (Table 1). Considering coffee seedlings inoculated with AMF and with low doses of P in the soil, there was no positive effect on increased leaf area Collozi-Filho, 1986).
This result is favorable for the plant since it provides benefits such as a greater initial growth of the aerial part, greater photosynthetic rate and larger stem diameter among others. Studies show that AMF promoted increases in the chlorophyll contents in leaves of coffee (Tristão;Andrade;. The results of this study indicate that the species of inoculated AMF affects the response of the plant, corroborating with other studies with AMF in symbiosis with plants ( Saggin-Júnior et al., 1995;Moreira et al., 2019).
The total chlorophyll content was higher in seedlings inoculated with R. clarus and with R. clarus and G. margarita, differing significantly from other the inoculated treatments (Figure 1).  Shoot dry mass (SDM), root dry mass (RDM) and total dry mass (TDM) were influenced by fungi and depended on the studied clone (Table 2). For Clone 2, SDM did not differ between the treatments. As for Clone 1, seedlings inoculated with AMF in F2, F3 and F5 showed increases of SDM when compared with the control treatment. When comparing the studied clones, Clone 1 presented higher SDM values than Clone 2 ( Table 2).
The root dry mass of Clone 2 did not differ between treatments. As for Clone 1, seedlings inoculated with FMA in F1, F2, F3, F4 and F5 showed increases when compared with the control treatment. Between the studied clones, Clone 1 presented higher values than Clone 2 for the treatments inoculated with FMA in F1, F2, F3 and F5 (Table 2).
For total dry mass, Clone 1 inoculated with AMF in F1, F2, F3, F4 and F5 showed increases when compared with the control treatment (Table 2). Between the studied clones, Clone 1 presented higher values than Clone 2 for the treatments inoculated with FMA in F1, F2, F3 and F5 ( Table 2).
The main effects of the studied fungi influenced the accumulations of N and P. On average, the accumulations of N on both clones were greater in seedlings inoculated with AMF in F3, F4 and F5 when compared with noninoculated seedlings (control). As for the accumulation of P, the values were higher in seedlings inoculated with AMF in F2, F3, F4 and F5 (Figure 2). The accumulation of Ca considering the averages of both studied clones was greater in seedlings inoculated with AMF in F5 (Figure 2). Accordingly, the parameters of vegetative growth and SDM, RDM and TDM promoted satisfactory gains with the inoculation with AMF in Clone 1 (Table 2). Siqueira et al. (1995) obtained similar results in studies using coffee plants inoculated with AMF alone or in combinations, which presented higher values for SDM and RDM when compared with the control treatment.
Other studies corroborate with the results obtained in the present study, in which coffee plants inoculated with several species of AMF increased the absorption of N and P (Hodge;Campbell;Fitter, 2001). Similar results for the assessed nutrients were observed for C. arabica L. inoculated with AMF isolated or in combinations (Fonseca et al., 2019).
Colonization was greater in seedlings inoculated with the mixture differing significantly from the other inoculated treatments. The results of the percentage of colonized roots were satisfactory, since non-inoculated plants (control) failed colonization, and treatment F5, which consisted of three genes or species of AMF, showed the highest percentage of colonization ( Figure 3). The percentage of colonized roots, the content of chlorophyll and shoot dry mass were influenced only by inoculation with AMF, being observed increments in these variables in the presence of AMF when compared with the control treatment ( Figure 3) regardless of the studied clones.
Multiple studies with different genetic materials of coffee plants inoculated with AMF showed variations in the percentage of colonized roots with environmental influence by various biotic and abiotic factors Flores-Aylas et al., 2003;Costa et al., 2005;Berbara;Fonseca, 2006;Machineski;Balota;Souza, 2011;Albán;Guerrero;Toro, 2013;Mustafa et al., 2016;Bertolini et al., 2020). Another important point to be explored is how the species and genes of AMF used in the controlled inoculation. Moreira et al. (2019) studied Gigaspora margarita and Rhizophagus clarus and observed an increase to shoot dry mass and root system when compared with the noninoculated treatment. Therefore, the species of AMF affect positively the answer of the plant and present a positive point regarding programs of controlled use of AMF. It is necessary the use of effective inoculants that are adapted to the soil, climate conditions and culture management. Tristão, Andrade and Silveira (2006), evaluating Yellow Catuaí seedlings IAC 62 inoculated with AMF in different substrates, observed a wide variation in the percentage of roots colonized with values ranging from 3.1 to 39.1%. The values are similar to the results of the present study.
Therefore, the parameters of growth in height, stem diameter, shoot and root dry mass, total leaf area, chlorophyll content and total deposits of N, P and Ca were positively affected by inoculation of AMF in at least one of the studied clones, showing greater effects when compared with groups of non-inoculated seedlings. This fact observed on the results obtained in this study where significant positive correlation was found between mycorrhizal colonization, root dry mass, total dry mass and accumulation of Ca (Table 3) in Clone 1 contributed to the development of the plant due to the increased absorption of nutrients, especially Ca. On the other hand, Clone 2 did not present this effect.  Other authors have observed similar results for cultivars of C. arabica L. inoculated with AMF isolated or in combinations (Tristão;Andrade;Sammaurina et al., 2020). When plants are colonized with AMF, they generally do not show signs of deficiency. Moreover, they present better field adaptation aspects, such as survival and initial growth (Surpano et al., 2015;Prates Junior et al., 2019). There, the effectiveness of using AMF for the formation of coffee seedlings in nursery environments can be transferred to a most advanced phase of development of the coffee plants. Furthermore, another point to be noted is the quality and mineral nutrition of plants, providing greater vigor and greater productivity of coffee plants.

CONCLUSIONS
The use of AMF improved the development of micro-propagated seedlings of Coffea arabica L. in the phase of acclimatization. The studied clones presented different behaviors when inoculated with AMF. Clone 1 (red fruiting Catucaí) showed better ability to be used in the production of micro-propagated seedlings using AMF.

ACKNOWLEDGMENTS
The authors acknowledge FAPEMIG for the financial support, CNPq for the productivity scholarships Table 3: Clone correlation between the percentage of colonization, height, stem diameter, shoot dry mass, root dry mass, total dry mass, leaf area, chlorophyll content and accumulation of N, P and Ca for seedlings of coffee with and without the use of arbuscular mycorrhizal fungi on a greenhouse experiment, Varginha MG.