Evaluation of the effectiveness of some mycorrhizal fungi isolates against charcoal rot disease

The sunflower plants are attacked by serious seed and soil-borne pathogens including charcoal rot disease that caused by Macrophomina phaseolina. This disease has serious damages to sunflower crop. This study aimed to assess the efficacy of Arbuscular mycorrhizal fungus against charcoal rot disease as fungicide alternative. Morphological and molecular identification was done, using universal primers for molecular identification. Finally, a greenhouse experiment was conducted, and the length and weight of the plant shoot and root as well as disease incidence and severity percentages were estimated for the treatments infected with the M. phaseolina pathogen. The results showed that molecular identification indicates their subordination to Claroideoglomus etunicatum and Funneliformis mosseae. The greenhouse experiment showed that combined inoculation treatments (C. etunicatum + F. mosseae and C. etunicatum + F. mosseae + M.phaseolina) had the highest averages of length and weight of the plant shoot and root in the non-infected and pathogen-infected (M. phaseolina) treatments. F. mosseae and F. mosseae + M.phaseolina treatments had better effects on plant growth. M. phaseolina pathogen had suppression effects than the C. etunicatum inoculum (C. etunicatum and C. etunicatum + M. phaseolina treatments). The disease incidence and severity percentages decreased significantly in the inoculated plants than in non-inoculated plants. F. mosseae inoculum showed more efficiency in reducing DI and DS than C. etunicatum inoculum, while adding both, resulting in an extra significant reduction. The combined inoculation, which functions as a biofungicide and a biofertilizer, presented a viable biocontrol technique for crop protection. Such the AM fungi treatments are considered good and biological safe. The symbiotic relationship of AM fungi with plants can also be utilized to gain successful management of agricultural against soil-borne diseases for getting rid of the negative effects of pesticides.


Background
The sunflower (Helianthus annuus) is one of the most important oil seed crops as it contains high-quality edible oil (40-50% by weight) and protein (15-21% by weight) (Haj Sghaier et al. 2023).Diseases are serious threats to the sunflower crop all over the world.Charcoal rot caused by Macrophomina phaseolina is of prime importance in reducing crop yield, especially in arid regions of the world.It has been estimated that the disease can cause an average annual loss of 12% in yield, from nearly 12 million hectares of world land (Ijaz et al. 2013).Macrophomina phaseolina is an important soil-borne pathogen that infects many economically important crops such as sunflower, maize, cotton, soybean, and sesame (Adel-Kaderet et al. 2010).Chemical fungicides are used to control the M. phaseolina pathogen.However, these chemicals are not eco-friendly; hence, an alternative method is needed to manage this disease.Recently, soilborne diseases have been controlled by utilizing certain beneficial microorganisms that are indigenous to the plants' rhizosphere (Kumar et al. 2014).Various microorganisms have been used effectively, such as Bacillus spp., Trichoderma species, and Arbuscular mycorrhizal (AM) fungi, as biological control agents for several pathogens (Weng et al. 2022).These bioinsecticides provide a better option than chemicals due to their safety for humans and the environment and lower cost (Adekunle et al. 2001).AM fungi, which are widely spread in soil environments, can develop symbioses with more than 80% of terrestrial higher plants and enhance the plants' nutrition (Weng et al. 2022).The fungi have been broadly used as biological control agents to control numerous phytopathogenic fungi (Lin et al. 2021).AM fungi can organize secondary metabolite formation in the host plants by altering the plant roots' morphology or anatomical structure, developing the rhizosphere environment characteristics (chemical and physical), competing with the pathogens for infection space and photosynthetic products, and activating defense systems and disease resistance in plants (Aseel et al. 2019).According to Sudhasha (2020), the chemical balance of mycorrhizae inhibits growth and reproduction of pathogenic fungi.They found out that G. intraradices prevented the growth of F. oxysporum.Aguk et al. (2018) found that the disease incidence and index of Ralstonia solanacearum treatments inoculated with G. mossie and G. rhizogenes were reduced by 49.8 and 9.7%, respectively, than the treatment not inoculated with AM fungi (the control).
As a result, the employment of AM fungi is regarded as a technique in sunflower plant improvement.It was necessary to use AM fungi that were adapted to the region's environmental conditions.Thus, the study's goal was to isolate and identify AM fungi and assess their efficacy on sunflower plant growth and the incidence of charcoal rot.

Soil samples
An extensive field survey was carried out (in August 2021) to randomly collect the sunflower (Helianthus annuus) rhizospheric soil samples.About 500 g of 30 rhizospheric soil samples near the root zone (depth 15-20 cm) was collected in sterile polyethylene bags.The soil samples were transferred to the laboratory in an icebox, where they were kept at 4 °C until use.

Monosporal culture
A variety of techniques can be used to separate monosporal cultures from the soil.The wet-sieving and decanting technique was used to isolate Arbuscular mycorrhiza (AM) fungi spores (Gerdemann and Nicolson 1963).After filtration, spores of different sizes were picked up, while observing under a microscope.Then, they were observed under a microscope after being mixed with a drop of lactophenol blue (phenol, lactic acid, aniline blue dye, and glycerol) on clean slides.

Microscopic identification
Spores' morphology serves as the basis for the identification of AM fungi because the hyphae and the organs, like vesicles and arbuscules, are not species-specific.AM fungal spores were observed microscopically, and identifications, at the genus level, were performed by examining diagnostic characteristics such as spore size, shape, color, wall, and type of hyphal attachment (Oehl et al. 2011).

Extracting of DNA
DNA was extracted from AM spores (morphologically identical) by crushing the spores with 40 µl of Tris-EDTA (TE) and heating them at 95 °C for 10 min in the presence of 10 µl of Chelex-100 (Dong and Zhao 2006).

PCR technique
AM fungi species were identified using the PCR technique and the universal primers ITS1 F and ITS4 R. The PCRs were achieved in a total volume of 25 μl; 5 µl DNA, 5 μl of Taq PCR PreMix (Intron, Korea), 1 µl of each primer (10 pmol), and then, distilled water was added into the tube (13 μl).The thermal cycler was programmed as shown in Table 1.The results were obtained by the software provided with the device.Electrophoresis was performed to examine the amplification products on a 1.5% agarose gel stained with ethidium bromide (EtBr).

Collection of Macrophomina phaseolina
An intensive survey was conducted (during the summer of 2021) on the incidence of charcoal rot in different locations of sunflower fields.Twenty samples of sunflower plants showing typical symptoms of charcoal rot were collected from the various hosts.The roots were transferred to the laboratory, rinsed carefully with tap water, surface-sterilized with 0.1% sodiumhypochlorite solution, and washed three times with sterilized distilled water before being cut into small pieces and air-dried.The root pieces were incubated on PDA (potato dextrose agar) Petri plates in the dark for seven days at 28 ± 2 °C.The arisen fungal colonies (M.phaseolina) were examined and then maintained as pure cultures on PDA slants, using the single hyphal tip technique (Singh and Srivastava 1988).Finally, M. phaseolina fungal were identified by the PCR technique and the primer pair ITS5 (5′-TCC GTA GGT GAA CCT GCG -3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC -3′) based on amplifying the genomic DNA region of the fungi (Integrated DNA Technologies company, USA) (White et al. 1990).

Pot culture
Sorghum (Sorghum halepense L.) and alfalfa (Medicago sativa L.) host species were used as trap plants for the mass production of soil-based AM spores inocula (Kapoor et al. 2007).Sterilized pots were loaded with 3 kg of soilrite: soil (1:3) sterilized mixture and then covered with 100 spores of AM inoculum per 3 kg of soilrite, for both C. etunicatum and F. mosseae isolates separately.Fungi species have an inoculum density of 100-105 spores per 3 kg of soil.Sorghum and alfalfa seeds were sanitized with HgCl 2 (0.01%) for two min and then washed three times with sterilized distilled water before being transferred to the pots.Twentyfour pots were used: 12 pots for each plant species of which the six ones were for each fungus species.The pots were kept in a greenhouse under controlled conditions for 60 days.Of the nine plants, three were chosen at random and pulled from each pot.Three plants were chosen at random and pulled from each pot.Root samples were washed with sterile water, then sterilized with sodium hypochlorite (NaOCl, 2%) for 5 min, and rewashed with sterilized distilled water.After staining with Trypan blue (Philips and Hayman 1970), the samples were cut into 1-cm segments, and AM colonization was investigated according to the following equation:

Soil preparation
The soil was collected from the fields, sieved through 2mm after air-drying, and then autoclaved for 1 h at 121 °C (Bashan et al. 1995).The properties of the soil are presented in Table 2.

Inoculums preparation
Mass-multiplied AM fungi species that were obtained by pot culture were used as inoculums.One hundred grams of roots plus soil, which contained 100 spores per 1 g, was used as inoculums for C. etunicatum and F. mosseae treatments.M. phaseolina isolate was grown on local millet seeds, Panicum miliaceum L. The seeds were washed well and moistened for 6 h and then dried at laboratory  Nitrogen (mg kg −1 ) 2.12 Phosphorus (mg kg −1 ) 10.3 Organic matter (g kg −1 ) 1.11 Calcium phosphate (g kg −1 ) 140 Calcium carbonate (g kg −1 ) 157 Sand (g kg −1 ) 510 Silt (g kg −1 ) 290 Clay (g kg −1 ) 200 Texture Sandy clay loam temperature.One hundred grams of millet seeds was placed in a 250-ml flask, a little water was added, and it was autoclaved.After cooling, the flasks were inoculated with a M. phaseolina isolate and incubated at 25 ± 2 °C for 10 days, with shaking every 2-3 (Dewan and Sivasithanparm 1988).

Greenhouse pots preparation and cultivation
Sterile pots were filled with 8 kg of autoclaved soil.Sunflower seeds were planted (10 seeds per pot) after applying 100 g of mycorrhizal inoculum at 3-4 cm depth.
Pathogen inoculum was inoculated around the plants' roots after 20 days of germination.

Plant growth assessment
Plant growth was assessed after 45 days from germination by estimating the length and weight of each plant shoot and root.Air-drying plant shoots were crushed for digestion (Gresser and Parsons 1979), and the concentrations of calcium (Ca), potassium (K), and magnesium (Mg) were determined using a flame photometer, while phosphorus (P) was determined using a spectrophotometer (Tandon 1995).

Disease incidence and disease severity
Percentages of disease incidence (DI) and disease severity (DS) were calculated for the treatments infected with the M. phaseolina pathogen.DI, the infected leaves percentage, is calculated according to the following formula: DS was evaluated on a 5-degree scale (from 1 to 5) according to symptom severity, wilt, develop a grayblack discoloration, external discoloration on the basal part of the stem, and depth 1 = healthy, 2 = lesion on stem only, 3 = 25% plant symptomatic, 4 = 26-50% plant symptomatic and 5 ≥ 50% of the plant symptomatic (Shokes et al.1996) as presented in the following formula:

Statistical analysis
The experiment was carried out in a complete random design (CRD) in vitro condition, and data were analyzed for variance (ANOVA) using the SAS software version (9.1).The means were compared using a 0.05 least significant difference (LSD).

Microscopic identification
Sunflower rhizosphere soil from the Al-Alam area, in Saladin governorate-Iraq, showed the presence of various types of AM spore populations.Spores' morphology serves as the basis for the identification of AM fungi because the hyphae and the organs, like vesicles and arbuscules, are not species-specific.AM fungal species were identified at the genus level after their spores were isolated from soil samples and adopted the taxonomic key mentioned in Oehl et al. (2011).The morphological identification showed that the spores were related to two species after matching its characteristics with the key, which depends on the spore's color, size, external shape, contents, number of walls, thickness of the walls, and type of hyphal attachment.
The results showed that 17 (56.7%)samples seemed to be related to Funneliformis spp.and 13 (43.3%)samples seemed to be related to Claroideoglomu spp.Spores collected from soil often deteriorate, which is why they can be used only for tentative identification at the genus level.Therefore, molecular identification was done for the most abundant and similar in morphological for spores obtained from the 30 samples.The spores of mycorrhizal fungi spread in many soils, but there is a difference in the species and number of spores due to the type of host plant, the genus of the fungus itself, the physical, chemical, and fertility properties of the soil (Lynch 2011).

Molecular identification of AM fungi
In general, the ITS region assay is a commonly used method for the diagnosis of fungi, AM fungal spores were classified, and typical spores were chosen.The DNA was extracted from spores, and then, PCR analysis was performed.An approximately 650 bp of ITS DNA fragment was successfully amplified from AM fungi species (Fig. 1).AM fungi species showed similarity rate, which

Molecular identification of Macrophomina phaseolina
The collected fungal strains from sunflower plants showing typical symptoms of charcoal rot were identified as M. phaseolina by the production of several individual sclerotia on the Petri plates containing PDA.The DNA was extracted from spore samples, and then, PCR analysis was performed.An approximately 550 to 700 bp of ITS DNA fragment was successfully amplified from all M. phaseolina (Fig. 2).M. phaseolina isolate was recorded at the gene bank, NCBI, No: OQ215734.1.

Pot culture
The percentages of F. mosseae spores' colonization were higher than those of C. etunicatum spores in both Sorghum and alfalfa host plants.The colonization of F. mosseae spores was 100% and 95% in Sorghum and alfalfa host plants, respectively, while the colonization was 80% and 70% for C. etunicatum spores in Sorghum and alfalfa host plants, respectively (Table 3).Sorghum plants showed higher C. etunicatum and F. mosseae colonizations than alfalfa plants, suggesting that the Sorghum host species is the preferable plant trap for the mass production of soil-based AM spore inocula.
AS shown in Fig. 3, F. mosseae and C. etunicatum fungi colonizing spores in the Sorghum host plant.The colonization of AM fungi and the subsequent production of spores depend on the host type and increase with the infection period (Suada et al. 2018).Studies discovered that mycorrhizal colonization and spore density did not significantly correlate with soil pH, Mg, Ca, Al, K, or organic matter levels, but only with P content.The highest colonization rates take place at low or absent P fertilization levels (Santos et al. 2017).

Greenhouse experiment
The greenhouse experiment treatments are depicted in Figs. 4 and 5 as illustrated; shoot length (A), root length (B), shoot fresh weight (C), and root fresh weight (D) of sunflower plants after 45 days from germination.

Shoot length
The average of sunflower shoot length after 45 days from germination recorded a significantly the highest value when both C. etunicatum and F. mosseae inoculums were added (C.e + F.m), which was 102.25 cm.The addition of F. mosseae and C. etunicatum separately (F.m and C.e

Root length
The highest length of roots was recorded by C.e + F.m treatment (13.96 cm), followed by F.m (13.92 cm), and then C.e treatments (12.63 cm) with non-significant differences, but the three treatments showed a significant increase from the control (C) treatment (11.16 cm).In the pathogen-infected (M.phaseolina) treatments, the binary inoculation treatment (C.e + F.m + M.p) showed the highest root length (12.48 cm), which differs significantly from the single inoculation.The F.m + M.P treatment (11.02 cm) had greater significant effect on pathogen suppression than the C.e + M.P treatment (8.87 cm), but both treatments increased plant root length significantly as compared to M.p treatments (5.98 cm).

Shoot fresh weight
The C.e. + F.m. treatment recoded the highest average of shoot fresh weight (79.1 g), followed by the F.m. (77.21

Root fresh weight
The combined inoculation treatment (C.e + F.m) had the highest average of fresh root weight (5.88 g), which was significantly different from the other treatments.Single inoculation (C.e and F.m treatments) showed a significant increase in the fresh root weight (5.21 and 5.3 g) than the control (C) treatment (4.45 g), whereas in the pathogen infection treatments, (C.e + F.m + M.p) and (F.m + M.p) treatments (5.4 and 5.08 g, respectively) revealed a significant increased in root fresh weight from the rest treatments, followed by (C.e + M.p) treatment (3.89 g), which also showed a significant increase from M.p treatment (3.07 g).

Ca, K, Mg, and P concentrations
The concentrations of Ca, K, and Mg were determined in the plant shoots after 45 days from sunflower germination (Fig. 6).The results showed that the combined inoculation treatment (C.e + F.m) recorded the highest Fig. 4 The greenhouse experiment treatments average of these element concentrations, followed by the F.m treatment, then C.e, with a significant difference between them and a significant increase than the control treatment (C).In the pathogen-infected treatments, the combined inoculation by (C.e + F.m + M.P) showed a significant increase at the concentrations of all the tested elements.But the significance of the single inoculation's treatments varied.The (F.m + M.p and C.e + M.p) treatments had significantly decreased Ca concentration values than the (C.e + F.m + M.p) treatment, with a significant difference between them and a significant increase than the M.p treatment.K concentration increased significantly in the (C.e + F.m + M.p) treatment.(F.m + M.p) treatment showed a significant increase from M.p treatment but a non-significant increase from C.e + M. p treatment, which in turn showed a non-significant increase from M.p treatment.Mg concentration recorded a significant increase value in (C.e + F.m + M.p) treatment, followed by (F.m + M.p) then (C.e + M.p) treatments that showed a significant difference between them, while (C.e + M.p) treatment revealed a non-significant difference from M.p treatment.The concentration of P recorded a significant increase in the combined treatment (C.e + F.m + M.p).The treatments (F.m + M.p and C.e + M.p) did not show a significant difference between them, but they differed significantly from the M.p treatment, and the better effect of F. mosseae inoculum on plant growth and M. phaseolina pathogen suppression.

Disease incidence and severity
AM fungal inoculated plants were less influenced by the presence of M. phaseolina pathogene, as a result of the F. mosseae and C. etunicatum fungal promoting roles in the plant growth enhancement (Fig. 7).The disease incidence percentage decreased significantly in the inoculated plants than in the non-inoculated plants (M.p treatment), which was 93.00%.F. mosseae (51.40%) had more effect than C. etunicatum inoculum

Discussion
DNA barcoding is so useful for daignosis of fungal types.The internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene bunch are utilized as the fungal barcode and the major advantage of using ITS is making it possible to amplify the gene from small amounts of biological specimens (Abarna and Vishnupriya 2022).The molecular identification was carried out by DNA barcoding using the ITS region sequencing.
The ITS rDNA sequences were compared with those in the databases using NCBI-BLAST.AM fungi and the pathogenic fungus, M. phaseolina, were identified using DNA barcoding with an identity of 100%.The AM fungi have great influences, directly on enhance health plant and on the life on land.Therefore, molecular identification of AM fungi is becoming more crucial in scientific research and application.Depend molecular identification for AM fungi on the rRNA genes (Thilagar et al. 2018).In this study, the presence of the two types of AM  fungi (F.mosseae and C. etunicatum) was determined by PCR and followed by sequencing and recorded at the gene bank, National Center Biotechnology Information (NCBI).The previous results showed that the inoculation of plants with AM fungi, particularly F. mosseae, affected positively the length and weight of the vegetative plant parts and the roots.The presence of AM fungi inoculation reduced the pathogenic fungus's negative effect on the length and weight of the shoots and roots.The establishment of AM fungi in the host plant roots primarily reduced diseases caused by soil-borne pathogens (Dehne 1982), which could be attributed to AM fungi interacting with plant pathogenic organisms and improving the plants' defenses and resistance to M. phaseolina.Generally, in both non-infected and infected plants, combined inoculation treatments performed best.Numerous studies reported an increase in plant growth because of AM fungi colonization, arguing that increased nutrient and water uptake resulted in increased hormonal balance or photosynthetic production, resulting in increased plant biomass.Inoculating wheat, rice, and maize plants with AM fungi had a significant effect on their height, weight, and root colonization because this fungus may serve as a plant's natural fertilizer or biofertilizer (Bholay et al. 2018).The medicinal plant Solanum viarum that was colonized with AM fungi showed increased growth, plant height, and dry biomass, mainly due to an increase in the supply of phosphorus (Selvaraj et al. 2011).Huang et al. (2020) assessed the effect of five AM fungi species on walnut plant growth after 3 months of inoculation and found that Diversispora spurca-, Glomus etunicatum-, and Glomus mosseae-inoculated plants showed higher root length, surface volume, and area than non-inoculated plants.Also, D. spurca, Acaulospora scrobiculata, G. etunicatum, and G. mosseae significantly increased leaf transpiration, photosynthesis rate, and stomatal conductivity, while reducing leaf temperature and intracellular CO 2 concentrations.Consequently, they concluded that the positive role of AM fungi on walnuts was dependent on the fungus species.
These findings exhibited the potential contribution of mycorrhizal fungi in getting nutrients available, particularly immobile phosphorus, in the soil.The nutrient content analysis of sunflower indicated that F. mosseae had greater potential effect than C. etunicatum.Combined inoculation treatments revealed the highest concentrations of Ca, K, Mg, and P in the healthy and infected plants, but they decreased in the infected plants as compared to the healthy plants.AM fungus colonization can improve a plant's ability to utilize soil nutrients, particularly Ca, K, Mg, P, N, and Mn, and, as a result, increase plant growth (Weng et al. 2022).The increase in nutrient absorption area brought on by the action of widespread mycorrhizal hyphae in soils contributes to the increase in growth parameters carried on by mycorrhizal symbioses (Tarraf et al. 2017).Enhancements of plant growth and the uptake of nutrient by AM fungi inoculation were earlier elucidated by many researchers (Weng et al. 2022).P enhancement may be related to the organic acids and phosphate released by AM fungi (Nafady et al. 2019).
The colonization of AM fungi in plant roots increased their resistance to pathogenic microorganisms and decreased several diseases caused by soil-pathogenic organisms (Oyewole et al. 2017).Sunflower plants inoculated with AM fungi showed less damage from the pathogenic fungus.This signifies defense against pathogen attack on the root tissues, which may be due to competition between AM fungi and M. phaseolina for nutrients and space (Nafady et al. 2019).
Obtained results confirm a decrease in disease incidence and severity; this decrease may be due to competition for space and photosynthates between the AM fungus and the pathogen in the host plant, which is similar to the finding of Doley and Jite (2013).The AM fungus's effect on disease incidence and severity may be attributed to the fact that the MF affects the physiology of the host plant and positively activates the plant's resistance by creating or inducing a type of mechanical barrier against the invading pathogen (Wendimu 2022).

Conclusions
The symbiotic relationship of AM fungi with plants can be utilized to gain successful management of agricultural soils because of its significance in improving plant growth by increasing nutrient uptake and providing disease protection.The concept here is to use AM fungi as a natural, harmless, and effective alternative to pesticides and fertilizers.Sunflower plants inoculated with AM fungi showed remarkable improvements in comparison with healthy non-inoculated plants.In addition, with the help of AM fungi inoculations, the harmful effects of the M. phaseolina pathogen can be reduced in pathogenicinfected plants.

Fig. 3
Fig. 3 AM fungi colonizing spores in the Sorghum host plant; A Claroideoglomus etunicatum and B Funneliformis mosseae g) and C.e. (76.42 g) treatments with non-significant differences.These three treatments showed a significant increase from the control (C) treatment (64.1 g).The (C.e + F.m + M.p) treatment (64.42 g) showed a maximum shoot fresh weight in the infected treatments, followed by the F.m + M.P treatment (62.23 g), with non-significant differences, and the (C.e + M.p) treatment (49.98 g), with a significant difference.Nevertheless, when compared to the MP treatment (35.84 g), these three inoculation treatments resulted in a significant increase in shoot fresh weight.

Table 1
PCR program for ITS gene detection of the studied fungi

Table 2
The properties of greenhouse experiment soil

Table 3
The colonization percentage and spores number of Claroideoglomus etunicatum and Funneliformis mosseae in the Sorghum and alfalfa host plants