Evaluation of Some Chemical and Biological Fungicides for Controlling Stem Canker on Apricot Trees Caused by Neoscytalidium dimidiatum

doi:10.21203/rs.3.rs-4317360/v1

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Evaluation of Some Chemical and Biological Fungicides for Controlling Stem Canker on Apricot Trees Caused by Neoscytalidium dimidiatum

https://doi.org/10.21203/rs.3.rs-4317360/v1

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Neoscytalidium dimidiatum (Penz.) Crous & Slippers has been causing significant damage to apricot trees in Turkey in recent years. This disease can lead to various problems in apricot trees, including dieback, the formation of cankers, necrosis in vascular tissues, gumming, and ultimately the death of the host. This study was conducted in 2021–2022 in the region with the highest apricot production in the world, Malatya, Türkiye. This research aims to develop a control strategy against N. dimidiatum, which poses a destructive threat to apricot trees. In the study, the effectiveness of 10 chemical and 2 biological fungicides was tested in vitro and under field conditions to manage N. dimidiatum. While the effectiveness of chemical fungicides was assessed both in vitro and under field conditions, the effectiveness of biological fungicides was tested only under field conditions. Given the absence of a study conducted under field conditions for the controlling of N. dimidiatum, this aspect of the research is groundbreaking.

In vitro experiments were performed in three replicates for each of the chemical fungicides. In the conducted in vitro experiments, most of the tested chemical fungicides, except for metalaxyl-m + acibenzolar-s-methyl (10.46%), effectively inhibited the mycelial growth of N. dimidiatum. In terms of inhibiting the mycelial growth of N. dimidiatum, the most effective fungicides were ranked as follows: tebuconazole (100%), cyprodinil + fludioxonil (99.43%), azoxystrobin + difenoconazole (99.40%), and floupyram + tebuconazole (99.26%). Chemical fungicides that exhibited high efficacy under in vitro conditions also proved to be effective in field trials. Among these fungicides, azoxystrobin + difenoconazole, floupyram + tebuconazole, and tebuconazole were identified as the most effective fungicides both before and after artificial inoculation. Cyprodinil + fludioxonil showed high efficacy when applied before inoculation but was not effective when applied after inoculation. In contrast to the chemical fungicides, the commercial Bacillus subtilis and Trichoderma harzianum Rifai KRL-AG2, which were exclusively examined in field studies, did not display significant effectiveness against N. dimidiatum.

Apricot

biological control

disease control

fungicide

Neoscytalidium dimidiatum

stem canker

Worldwide, approximately 4 million tons of apricots were produced on around 5.6 million decares of land. Turkey holds the top position in the world ranking, accounting for 23.6% of this production (FAO, 2021). Despite being a major player in global apricot production, Turkey's apricot production faces significant losses, particularly due to some fungal diseases (Kural & Erdiler 1995; Çığşar, 1998; Özgönen & Erkılıç, 2001; Oksal et al., 2020b). Neoscytalidium dimidiatum, among these fungi, is a significant fungal pathogen responsible for causing cankers in a wide geographical and host range, including apricot, almond (Prunus dulcis), mango (Mangifera indica), pitahaya (Hylocereus undatus), Citrus, Musa, Populus, Ficus species, and royal poinciana (Delonix regia) in countries such as Australia, China, Egypt, Niger, Tunisia, the United States, and the United Arab Emirates (Reckhaus, 1987; Nouri et al., 2018; Al Raish et al., 2020). In recent studies conducted in Turkey, N. dimidiatum has been reported as a harmful pathogen affecting various product groups, including apricot trees (Dervis et al., 2019; Dervis et al., 2020; Kurt et al., 2019; Oksal & Özer 2021; Oksal et al., 2020a; 2020b; Ören et al., 2020). N. dimidiatum species, which are becoming increasingly prevalent in Turkey, can act as plant pathogens, endophytes, saprophytes, and latent pathogens. Additionally, they are considered as causative agents of mycosis in humans and animals (Phillips et al., 2013; Mohd et al., 2013; Alizadeh et al., 2022; Türkölmez et al., 2019). This pathogen can lead to a range of detrimental effects on the plants it affects, including dieback, scorching, canker formation, necrosis in the woody tissues, leaf shedding, chlorosis, gumming, tip dieback, rot, and sometimes the complete death of the host. On apricot trees, in particular, it often causes destructive diseases such as shoot blight, dieback, and cankers (Oksal, 2020b).

Despite ecological concerns and health-related issues, the use of chemical fungicides continues to be the primary strategy for mitigating the threat of plant diseases (Saeed et al., 2016; Alwahshi et al., 2019; Kamil et al., 2018). Previous studies on the chemical control of N. dimidiatum in different product groups have reported the successful inhibition of the pathogen's mycelial growth or spore germination by chemical fungicides such as cyprodinil + fludioxonil, azoxystrobin + difenoconazole, tebuconazole, metiram, trifloxystrobin, iminoctadine, pyraclostrobin, azoxystrobin, hexaconazole, and difenoconazole + cyflufenamid (Lin et al., 2017; XiaoYong et al., 2018; Al Raish et al., 2020; Oksal et al., 2021). Recently, the use of biological control agents (BCAs) in disease management has become more widespread in addition to chemical fungicides. Biological control of N. dimidiatum is achieved, particularly using fungi and bacteria. For example, biocontrol agents such as Trichoderma harzianum and T. atroviride have been successful in reducing the growth of the pathogen. Furthermore, biokontrol bacteria like Bacillus amyloliquefaciens, Penicillium rolfsii, and B. subtilis have exhibited strong antifungal effects against N. dimidiatum under in vitro conditions (Ratanaprom et al., 2021; Wang et al., 2021). However, whether these biological control agents are effective against N. dimidiatum under field conditions remains unclear and requires further investigation.

This study was conducted to assess the potential effectiveness of chemical and biological fungicides against N. dimidiatum. The primary aim of this study is to evaluate the effectiveness of chemical fungicides against N. dimidiatum under in vitro conditions and highlight their performance in field studies, particularly focusing on the most efficient chemical fungicides in both scenarios. Additionally, this study aims to reveal, for the first time, the effectiveness of biopreparations with biocontrol potential under field conditions, as documented in the literature.

Plant material and fungal ısolates

The fungal material for the study was isolated from Hacıhaliloğlu and Kabaaşı apricot varieties. These plant materials were obtained from apricot trees showing symptoms of drying in Battalgazi, Yesilyurt, Darende, Kale, Akcadag, Hekimhan, Yazıhan, and Puturge districts in the 2018–2019 period, within the scope of Inonu University Project No: FBG-2018-977 (Fig. 1). The N. dimidiatum isolate (ITS MH861121, EF1-α KF531795; BT2 KF531796; LSU DQ377922), identified as the most virulent in the project's pathogenicity tests and molecular diagnosis, was selected for use in this study.

Fungicides

In vitro experiments involved testing the commercial formulations of 10 chemical fungicides for their ability to inhibit the mycelial growth of N. dimidiatum (Table 1). The two biological preparations found to be the most effective in in vitro experiments were included, and their potential for preventing infections caused by N. dimidiatum under natural conditions was assessed (Table 2).

Table 1

Fungicides and doses used *in vitro* trials.
Active ingredient (a.i)	Commercial Name / Firm	Formulation*	Dose / 100 L water	Dose of application (µg /ml)
700 g/L Copper oxychloride	ZZ-Cuprocol/ Syngenta	SC	0- 100 ml-200 ml-300 ml	0, 700, 1400, 2100
Trifloxystrobin %50	Flint/Bayer	WG	0–10 g-12,5 g-15 g	0, 50, 62.5, 75
Tebuconazole %25	Folicur/ Bayer	WG	0–40 gr-60 gr-80 gr	0, 100, 150, 200
Floupyram 200 g/L + Tebuconazole 200 g/L	Luna Experience/ Bayer	SC	0–15 ml-25 ml-35 ml	0, 30, 50, 75
Cyprodinil %37,5 + Fludioxonil %25	Switch/ Syngenta	WG	0–30 gr-40 gr-50 gr	0, 112.5, 150, 187.5
%70 Thiophanate-Methyl	Quartette/Hektaş	WP	0–40 g-60 g-80 g	0, 280, 420, 560
%40 Metalaxyl-m + %4 Acıbenzolar-s-methyl	Bion/ Syngenta	WG	0–10 g-20 g-30 g	0, 40, 80, 120
Azoxystrobin 200 g/L + Difenoconazole 125 g/L	Quadris maxx/ Syngenta	SC	0–80 ml-100 ml-120 ml	0, 160, 200, 240
400 g/l Phosphorous Acid	Driftçe/Koruma	SL	0-300 ml-400 ml-500 ml	0, 1200, 1600, 2000
Azoxystrobin 250 g/L	Quadris/ Syngenta	SC	0–50 ml-75 ml-100 ml	0, 125, 187.5, 250

*WG: Water-dispersible granule, WP: Wettable powder, SC: Water-soluble concentrate, SL: Soluble concentrate.

Table 2

Fungicides and doses used in the field trials.
Active ingredient (a.i)	Dose of application
Tebuconazole %25	40 gr
Cyprodinil %37,5 + Fludioxonil %25	40 gr
Floupyram 200 g/L + Tebuconazole 200 g/L	25 ml
Azoxystrobin 200 g/L + Difenoconazole 125 g/L	100 ml
%1.15 Trichoderma harzianum Rifai ırk KRL-AG2 (T 22) 400 milyon conidi/g	60 gr
Bacillus subtilis 13,4 g/L	1500 ml

Determination of the effect of fungicides on mycelial growth

Potato Dextrose Agar (PDA, Neogen) was used in Table 1 to determine the levels of inhibitory action of the fungicides listed on the mycelial growth of the N. dimidiatum isolate. In vitro experiments considered the usage conditions in registered products when determining the doses of fungicides, and experiments were conducted with both lower and upper doses (µg/ml) of the recommended usage rates. The effectiveness of each active substance was tested with three doses and one control (see Table 1).

To obtain the targeted fungicide doses, new stock solutions at concentrations of 1,000 µg/ml and 100 µg/ml were prepared through dilution from initially prepared stock solutions at 10,000 µg/ml. The fungicides were dissolved in sterile distilled water when preparing the stock solutions. To achieve the desired fungicide doses, the required amounts were pipetted, autoclaved for sterilization, and then added to a sterile PDA medium that had been cooled to 45–50°C (Delen et al., 1984). Controls were prepared in a similar manner, but sterile distilled water was added instead of the fungicide solution. Care was taken to ensure homogeneity in the mixtures, and equal amounts of sterile water were added to all doses for this purpose. Subsequently, agar media containing the desired fungicide doses or no fungicide (control) were poured into sterile plastic petri dishes (Isolab, 90 x 100 mm) in equal amounts and left to solidify for 24 hours under UV light for sterilization. Fungal discs with a diameter of four millimeters were taken from the edges of 10-day-old fungal colonies of the N. dimidiatum isolate using a cork-borer and placed into the growth cultures with and without fungicide (application and control). The experiment was conducted with three replications in a randomized complete block design. Inoculated petri dishes were incubated in a lightless incubator set at 25 ± 1°C for four days. Four days after incubation, the colony growth and diameter measurements of the N. dimidiatum isolate were evaluated.

The data obtained from the experiment were subjected to analysis of variance using the SPSS statistical software package, and the differences between the means were determined using the Duncan test (P ≤ 0.05). The percentage efficacy of fungicides was calculated using the Abbott formula, based on the colony diameter in the control: [(control - treatment) / control) × 100)] (Abbott, 1925).

Field trials

To determine the effectiveness of four chemical fungicides found to be the most effective in in vitro experiments and, in addition, two biopreparations not included in the in vitro study, in controlling infections caused by N. dimidiatum during the growth process of two-year-old grafted Hacıhaliloğlu apricot saplings, the recommended dosage ratio in registered products was used for biopreparations, as shown in Table 2. Fungicides were applied in the following sequence: 48 hours after inoculation (post-inoculation treatment) and 48 hours before inoculation (pre-inoculation treatment).

For post-inoculation treatment, mycelial discs (4 mm in diameter) obtained from PDA cultures of N. dimidiatum were inoculated into wounds made in the stem section, with the growing part facing inward, and then sealed with parafilm to prevent rapid dehydration (Úrbez-Torres, 2014). In control plants, only discs containing sterile PDA were used (Fig. 2A). Inoculated saplings were planted in pots containing a mixture of 1/3 seedling soil, 1/3 composted animal manure, and 1/3 sand. After these procedures, each pot was left for 48 hours of incubation under natural conditions. At the end of the incubation period, application of the fungicides listed in Table 2 and foliar application was performed by spraying. Only pure water was applied to control plants.

For pre-inoculation treatment, wounds made in the stem part were initially treated with the fungicides listed in Table 2, followed by foliar application. Control plants received an application of sterile distilled water. After 48 hours of application, pathogen inoculation was carried out on the wounds made on the plant stems using the fungal disk method as described by Úrbez-Torres et al., (2014) (Fig. 2B).

The experiment was regularly monitored for a period of 21 days, as determined in preliminary studies. At the end of the 21-day period, tissue samples were taken from the re-isolation points of the inoculation and placed on petri dishes containing streptomycin sulfate (150 mg/L). The relationship between the color change and the pathogen responsible for the condition was established (Eskalen et al., 2007; Mutawilla, 2011; Kotze et al., 2011). In the evaluations, the inoculation site and bark tissue of each test plant were scraped with a scalpel, and the length of the darkened area was measured with a ruler after the scraping process. Following the measurements, the percentage efficacy of fungicides was calculated using the Abbott formula, based on disease development in control plants: [(control - treatment) / control) × 100)] (Abbott, 1925).

Experimental design and statistical analysis

The experiment was conducted in a randomized complete block design with three replications, each containing four saplings. Controls were established in two different ways: one with only the pathogen present (positive control) and the other with no application (negative control) (Fig. 3). Data obtained from the experiment were subjected to analysis of variance using SPSS 17.0 (Ver.17.0, SPSS Inc., Chicago, IL, USA) software, and differences between means were determined using the Duncan test (P ≤ 0.05).

Determination of the effect of fungicides on mycelial growth

The effectiveness of the fungicides listed in Table 1 in inhibiting the mycelial growth of N. dimidiatum isolate was determined based on the lowest concentration that inhibited mycelial growth in the in vitro tests. After four days of incubation, colony growth and diameter measurements of N. dimidiatum isolate were performed.

When examining the fungicides based on their percentage of effectiveness in inhibiting the mycelial growth of N. dimidiatum isolate, tebuconazole, cyprodinil + fludioxonil, azoxystrobin + difenoconazole, and floupyram + tebuconazole were the most effective fungicides, with effectiveness ranging from 99–100%. These fungicides were followed by trifloxystrobin, thiophanate-methyl, and copper oxychloride with effectiveness between 83% and 85%, and phosphorous acid and azoxystrobin with effectiveness ranging from 74.8–60%. The fungicide containing metalaxyl-m + acibenzolar-s-methyl had a lower effectiveness of 10.4% in inhibiting the mycelial growth of N. dimidiatum isolate compared to other fungicides. The lowest effective doses of the most effective fungicides in the same statistical group were selected for field studies, including tebuconazole's first dose (40 g), cyprodinil + fludioxonil (40 g), azoxystrobin + difenoconazole (100 ml), and floupyram + tebuconazole (25 ml) (Table 3, Fig. 4).

Table 3

In vitro efficacies of fungicides (%) against *N. dimidiatum* isolate
Active ingredient (a.i)	Doses (ml/100L)	Average Colony Diameters (cm²)	Efficacy (%)
700 g/L Copper oxychloride	Control (0)	56,00	83,27
	100 ml	14,11
	200 ml	12,44
	300 ml	1,56
Trifloxystrobin %50	Control (0)	52,22	85,89
	10,0 g	7,56
	12,5 g	7,00
	15,0 g	7,56
Tebuconazole %25	Control (0)	45,28	100,00
	40 g	0,00
	60 g	0,00
	80 g	0,00
Floupyram 200 g/L + Tebuconazole 200 g/L	Control (0)	45,28	99,26
	15 ml	1,00
	25 ml	0,00
	35 ml	0,00
Cyprodinil %37,5 + Fludioxonil %25	Control (0)	58,67	99,43
	30 g	0,67
	40 g	0,33
	50 g	0,00
%70 Thiophanate-Methyl	Control (0)	58,67	83,71
	40 g	11,22
	60 g	9,22
	80 g	8,22
%40 Metalaxyl-m + %4 Acıbenzolar-s-methyl	Control (0)	58,78	10,46
	10 g	50,11
	20 g	58,67
	30 g	49,11
Azoxystrobin 200 g/L + Difenoconazole 125 g/L	Control (0)	42,94	99,40
	80 ml	0,78
	100 ml	0,00
	120 ml	0,00
400 g/l Phosphorous Acid	Control (0)	42,89	60,02
	300 ml	32,44
	400 ml	11,56
	500 ml	7,44
Azoxystrobin 250 g/L	Control (0)	43,11	74,83
	50 ml	12,11
	75 ml	10,56
	100 ml	9,89

Bakır oksiklorür, Trif: Trifloxystrobin %50, Teb: Tebuconazole %25, Flou + Teb: Floupyram 200 g/L + Tebuconazole 200 g/L, Cyp + Flud: Cyprodinil %37,5 + Fludioxonil %25, Thio: %70 Thiophanate-Methyl, M + Ac: %40 Metalaxyl-m + %4 Acıbenzolar-s-methyl, Azoxy + Dif: Azoxystrobin 200 g/L + Difenoconazole 125 g/L, Azoxy: Azoxystrobin 250 g/L

Field results

Two separate field trials were conducted to determine the effects of the fungicides listed in Table 2 on apricot saplings inoculated with N. dimidiatum. These trials were designed as post-inoculation and pre-inoculation fungicide applications. All data obtained from these trials were subjected to analysis of variance (ANOVA), and the effectiveness of the fungicides was determined. In the evaluations conducted 21 days after inoculation, it was found that fungicide applications (except for pre-inoculation Trichoderma application) were statistically significant compared to positive controls (P < 0.05). Brown-colored to black lesions of varying lengths were observed at the sites where the pathogen was inoculated, and these lesions were more pronounced in positive controls. In negative controls, color changes due to oxidation were observed, which was confirmed by the re-isolation studies (Fig. 5).

Effectiveness of the fungicides as post-inoculation treatments

In post-inoculation fungicide applications, the average lesion lengths in the vascular tissues were measured within a range of min. 29.0 ± 1.6 mm to max. 45.5 ± 2.0 mm. In the control group without fungicide application, the average lesion length was 50.0 ± 1.9 mm. When examining the effect of fungicides on lesion lengths in vascular tissues, azoxystrobin + difenoconazole (29.0 ± 1.6 mm) and floupyram + tebuconazole (30.2 ± 1.7 mm) were statistically significant (P < 0.05), followed by tebuconazole (38.6 ± 1.5 mm). Other fungicides were statistically in the same group as the control and did not have a significant effect on lesion lengths (Table 4). Azoxystrobin + difenoconazole and floupyram + tebuconazole were the most effective fungicides in terms of their curative aspect against N. dimidiatum, showing 41.8% and 39.5% efficacy, respectively, compared to the control. Floupyram + tebuconazole (26.6%) was the closest in terms of percentage efficacy, while other fungicides were not significantly effective compared to the control (Fig. 6).

Table 4

Determination of the post-inoculation effect of fungicides (%) used against *N. dimidiatum*
Active ingredient (a.i)	Mean Lesion Length (mm)	Efficacy (%)
Azoxystrobin 200 g/L + Difenoconazole 125 g/L	29,0 ± 1,6 a^*	41,8
Floupyram 200 g/L + Tebuconazole 200 g/L	30,2 ± 1,7 a	39,5
Tebuconazole %25	38,6 ± 1,5 ab	22,6
Bacillus subtilis 13,4 g/L	42,8 ± 2,0 b	14,3
Cyprodinil %37,5 + Fludioxonil %25	44,3 ± 2,0 b	11,3
Trichoderma harzianum Rifai KRL-AG2	45,5 ± 2,0 b	9,0
Positive control	50,0 ± 1,9 b	-

The average lesion lengths on inoculated apricot saplings. The effectiveness of applied fungicides against N. dimidiatum (%). * Significant differences exist between the mean values within each column as indicated by the Duncan's multiple range test (P = 0.05).

Effectiveness of the fungicides as pre-inoculation treatments

In pre-inoculation fungicide applications, the average lesion lengths in the vascular tissues were measured within a range of min. 20.9 ± 1.5 mm to max. 49.2 ± 1.4 mm. The control without fungicide application had an average lesion length of 50.0 ± 1.9 mm. When examining the effect of fungicides on lesion lengths in vascular tissues, tebuconazole (20.9 ± 1.5 mm) and cyprodinil + fludioxonil (22.0 ± 1.4) were statistically the most effective fungicides (P < 0.05). The fungicide azoxystrobin + difenoconazole (31.2 ± 1.6 mm) was the closest to them in terms of effectiveness. Bacillus subtilis (46.6 ± 1.6 mm) and Trichoderma harzianum Rifai KRL-AG2 (49.2 ± 1.4 mm) biopreparations were in the same group as the control and did not have a significant effect on lesion lengths (Table 5).

Tebuconazole (58.1%) and cyprodinil + fludioxonil (55.8%) were the most effective fungicides against N. dimidiatum in terms of protective efficacy, showing effectiveness of 58.1% and 55.8%, respectively, compared to the control. Following these fungicides in terms of effectiveness were azoxystrobin + difenoconazole (37.5%) and floupyram + tebuconazole (21.1%) in terms of percentage of effectiveness (Table 5). The other fungicides were not significantly effective in terms of percentage of effectiveness (Fig. 7).

Table 5

Determination of the pre-inoculation effect of fungicides (%) used against *N. dimidiatum*
Active ingredient (a.i)	Mean Lesion Length (mm)	Efficacy (%)
Tebuconazole %25	20,9 ± 1,5 a^*	58,1
Cyprodinil %37,5 + Fludioxonil %25	22,0 ± 1,4 a	55,8
Azoxystrobin 200 g + Difenoconazole 125 g	31,2 ± 1,6 ab	37,5
Floupyram 200 g/L + Tebuconazole 200 g/L	39,4 ± 1,5 b	21,1
Bacillus subtilis 13,4 g/L	46,6 ± 1,6 bc	6,6
Trichoderma harzianum Rifai KRL-AG2	49,2 ± 1,4 bc	1,5
Positive control	50,0 ± 1,9 bc	-

This study has demonstrated the efficacy of some fungicides in both in vitro and field conditions for controlling N. dimidiatum on apricot trees. Chemical fungicides, despite their potential adverse effects on health (Al-Tememe et al., 2019; Budzinski et al., 2018), are widely used due to their cost-effectiveness, quick action, long-lasting effects, stability, and ease of application (Kuai et al., 2017). In the in vitro part of the study for controlling N. dimidiatum, the tested fungicides aimed at inhibiting mycelial growth, with tebuconazole, cyprodinil + fludioxonil, azoxystrobin + difenoconazole, and floupyram + tebuconazole achieving the highest efficacy rates, ranging from 99–100%. Metalaxyl-m + acibenzolar-s-methyl, with a 10.4% efficacy rate, was the least effective fungicide in inhibiting the mycelial growth of N. dimidiatum (Table 3, Fig. 4). Lin et al., (2017) conducted a study on the main causal agent of pitaya cancer, N. dimidiatum, testing the efficacy of some fungicides against the pathogen in vitro. The results of their study revealed that cyprodinil + fludioxonil, azoxystrobin + difenoconazole, and tebuconazole fungicides were the most effective in inhibiting the mycelial growth of the pathogen. These fungicides were also identified as the most effective in our study. Similarly, XiaoYong et al., (2018) tested the effectiveness of pyraclostrobin, azoxystrobin, tebuconazole, and hexaconazole fungicides in an in vitro environment against pitaya cancer and reported that all fungicides were effective against the pathogen. The effectiveness of azoxystrobin and tebuconazole fungicides found in their study is consistent with our findings. In the United Arab Emirates (UAE), some fungicides were evaluated for their effectiveness in vitro and under greenhouse conditions against N. dimidiatum, which causes root cancer in royal poinciana (Delonix regia) trees. As a result of the evaluation, it was reported that azoxystrobin + difenoconazole (Amistrar® Top) and difenoconazole-based Cidely® Top (difenoconazole + cyflufenamid) fungicides, which are also included in our study, were among the most effective fungicides in inhibiting mycelial growth (Al Raish et al., 2020). In a recent study conducted by Oksal et al., (2021), the effect of 7 different fungicides at various dose series on the mycelial growth of N. dimidiatum was tested. In their study, all fungicides except phosphorous acid showed significant efficacy in inhibiting the pathogen's mycelial growth, with floupyram + tebuconazole, cyprodinil + fludioxonil, and tebuconazole standing out with 100% effectiveness. The results we obtained from our study are consistent with the findings of Oksal et al., (2021), except for the effectiveness of phosphorous acid.

In the field experiments, the lesions caused by N. dimidiatum in the vascular tissues of healthy apricot seedlings (Fig. 5) were similar to the symptoms observed under natural conditions, and this was confirmed by re-isolation. Our data are consistent with data from other studies where N. dimidiatum was artificially inoculated into other plant species (Al-Bedak et al., 2018; Nouri et al., 2018). In the growth process of apricot saplings of the Hacıhaliloğlu variety under field conditions, the most effective fungicides in preventing infections caused by N. dimidiatum were azoxystrobin + difenoconazole, floupyram + tebuconazole, and tebuconazole, both post-inoculation (Table 4) and pre-inoculation (Table 5). These fungicides, identified as the most effective in inhibiting mycelial growth in in vitro studies, also maintained their effectiveness under field conditions. Previous studies have also reported that these fungicides, either alone or in combination, are effective in preventing infections caused by N. dimidiatum (XiaoYong et al., 2018; Al Raish et al., 2020). Cyprodinil + fludioxonil showed high effectiveness when applied pre-inoculation (55.8%) but was not effective when applied post-inoculation (11.3%). The combination of cyprodinil + fludioxonil has been reported to be highly effective in controlling trunk diseases in grapes, which cause similar damage to N. dimidiatum. In these studies, immersing grapevine saplings in the cyprodinil + fludioxonil mixture before pathogen inoculation significantly reduced disease severity and positively affected growth parameters (Nascimento et al., 2007; Rego et al., 2009). In this respect, it can be concluded that the combination of cyprodinil + fludioxonil highlights the protective property of the fungicide. This observation aligns with the results of our study, demonstrating the effectiveness of these fungicides in controlling N. dimidiatum affecting apricot trees. Unlike chemical fungicides, the commercial biopreparations we included in our field study, B. subtilis and Trichoderma harzianum Rifai KRL-AG2, did not show significant effectiveness in preventing infections caused by N. dimidiatum, both pre-inoculation and post-inoculation (see Table 4 and Table 5). There have been some studies testing the effectiveness of BCAs against N. dimidiatum or different pathogens (AbuQamar et al., 2017; Rusmarini et al., 2017); however, these studies are generally of in vitro origin and do not evaluate their effectiveness in field conditions. Therefore, more research on the biological control of N. dimidiatum under field conditions is needed for accurate conclusions.

In conclusion, the increasing prevalence of N. dimidiatum requires the use of chemical control as a strategy to reduce its frequency and severity. Our study suggests that utilizing chemicals that have shown efficacy in both in vitro and field trials, taking advantage of their protective and curative properties, can be beneficial in the management of the disease and its subsequent control.

ACKNOWLEDGEMENTS

This work is supported with the project number TAGEM/BSAD/B/20/A2/P4/1677 by General Directorate of Agricultural Research and Policy of Ministry of Agriculture and Forestry

Research involving human participants and/or animals

This study does not include experiments with either human participants or animals.

Conflict of interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Evaluation of Some Chemical and Biological Fungicides for Controlling Stem Canker on Apricot Trees Caused by Neoscytalidium dimidiatum

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHOD

Plant material and fungal ısolates

Fungicides

Determination of the effect of fungicides on mycelial growth

Field trials

Experimental design and statistical analysis

RESULTS

Determination of the effect of fungicides on mycelial growth

Field results

Effectiveness of the fungicides as post-inoculation treatments

Effectiveness of the fungicides as pre-inoculation treatments

DISCUSSION

Declarations

References

Status:

Version 1