Morpho-Molecular Identification and First Report of Trichoderma Aggressivum is Causing Green Rot on White Button Mushroom in Iraq

The aim of this study was to morphological isolation and identification of fungus associated with the fruits and culture media of the white mushroom Agaricus bisporus. The results of isolation and identification from the sampling areas included Baghdad, Babylon, and Diwaniyah. Besides, Sulaymaniyah governorates showed the association of seven species of fungi associated the fruiting body and culture media of the white mushroom A. bisporus represented by the two pathogens Cladobotryum spp. and Trichoderma spp. As well as, the competitive fungi of A. bisporus culture medium, such as Aspergillus spp. and Penicillium spp. and Phoma sp. and Rhizopus spp., and Ulocladium sp. The most common pathogenic fungus was Cladobotryum spp. with a frequency of 56.2%, followed by Trichoderma spp., with a frequency of 54.1%, which appeared in samples from two sampling areas. Conversely, species T. aggressivum recorded a frequency of 42.5%, which is the first record in Iraq on the fruits and media of white fungus. The fungus Aspergillus spp. showed the existence percentage reached 65.5%, which is the highest among the competing fungi, followed by Penicillium spp., with an existence percentage of 26.6%. Then, Rhizopus spp., Phoma sp., and Ulocladium sp. with a frequency of 16.6%, 10.4%, and 10.4%, respectively. The results of the pathogenicity of the fungus isolate Trichoderma spp. indicated that all the isolates tested for the pathogenicity of the fruiting bodies of the white mushroom A. bisporus led to the infection of the fruiting bodies and their transformation into brown color and damage. The isolates of the fungus T. aggressivum (isolate 1 and isolate 2) recorded a disease severity of 60.0% and 60.0%, respectively. While, at a temperature of 4 °C, the isolates of the fungus T. aggressivum (1 and 2) recorded a disease severity amounted to 40.0% and 50.0%, respectively, compared to the control treatment without fungus, where the disease severity amounted to 0.0%. The results of the molecular identification of the DNA of the T. aggressivum, which showed the highest pathogenicity of A. bisporus fruiting bodies, using the specialized primer ITSI/ITS4, as it gave bands with a molecular weight of 600 bp. These findings were compared with the GenBank with a high similarity for the fungus T. aggressivum and it has been deposited in the Gen Bank with the accession number (OQ109172).


Introduction
The white mushroom (Agarics bisporus) is one of the foods that is distinguished by its delicious taste, distinctive flavor, and high nutritional value.It can be consumed in several different forms, including fresh, pickled, dried, canned, and other types of consumption according to the habits of the people in that country.The cultivation of the white mushroom A. bisporus has increased rapidly due to its economic returns and its low costs of cultivation, its abundant productivity.As well as, its nutritional and medicinal benefits [1][2][3][4][5][6].Fleshy -Edible Mushrooms have been known since ancient times as food, as they contain a high percentage of proteins, higher than most types of vegetables, as the percentage of proteins ranges from 10-40% based on the dry weight.China is the largest producer of A. bisporus white mushrooms among the top ten countries of global production [7,8].A. bisporus have long been used in ancient times to treat diseases like cancer, diabetes , antimicrobial and inflammatory throughout the world [9].The five most important fungi grown on a large commercial scale are A. bisporus, Pleurotus spp., Volvariella volvacea, Calocybe indica, and Lentinula edodes.In comparison, the most three important species are A.bisporus.and Pleurotus spp., Volvariella volvacea [10].The first outbreak of green mold disease for the first time in the eighties, according to reports about epidemics in Britain during the period 1985-1986.It causes a loss in the production of the white fungus A. bisporus estimated at 3-4 million sterling [11].This pathogenic fungus, known to be aggressive to the white mushroom A. bisporus, was also recorded in North America and caused very large losses estimated at 100% in production [12].Green Mold Disease was considered one of the biggest problems that could be encountered in the production of A. bisporus over several decades.Moreover, it is undoubtedly considered the most dangerous enemy and sometimes causes the loss of the entire crop [13].as the fungus Trichoderma spp. was diagnosed as causing this disease [14].However, [15].mentioned that there are nine species of fungus, Trichoderma spp., namely T. harzianum and T. spp.viride, T. hamatum, and T. koningii, T. polysporum, T. aureoviride, and T. aureoviride.piluliferum T, pseudokoningi T., and T. longibrachiatum [16].These species are considered a filamentous fungus and belongs to the Ascomycota, class Sordariomycetes, order Hypocreales, family Hypocreaceae.New species were found Trichoderma aggressivum f. europaeum and caused significant economic losses in A. bisporus production farms in Ireland and England [17], [18].The disease is easily distinguished by the growth of the pathogenic fungus in the form of dense white masses on the surface of the soil or compost.Then, it gradually changes to green after the formation and spread of spores with the appearance of brown discoloration on the fruiting bodies [19][20][21][22].Green mold disease often appears in development halls when the substrate is rich in carbohydrates and low in nitrogen [8].The fungus Trichoderma aggressivum is characterized by the formation of white colonies at the beginning of its growth on the culture medium (PDA).It gradually turns green, starting from the center of the colony, and after five days it turns into a dark green color, and the ideal temperature for its growth is between 25-30 °C.Likewise, it forms conidia smooth, oval, and green in color, formed on a cylindrical (phialide) shape borne on a conidiophore, confined between the branches of the conidiophores, which are adjacent or not [15,24,25].

Sampling
Samples were collected from several sites for the cultivation of the white mushroom Agaricus bisporus in Iraq , which are Baghdad governorate (Al-Wadaq farm/private farm), Babylon, Diwaniyah, and Sulaymaniyah governorates (private farms), with 31 samples.Emphasis was placed on samples that show symptoms of infection, represented by rotting, changing the color and shape of the fruiting bodies that appear in abnormal sizes and shapes, from the casing soil and the medium during the agricultural season 2021.Then, it was placed in polyethylene bags and brought to the Plant Diseases Laboratory of the Plant Protection Department / College of Engineering Sciences Agricultural / University of Baghdad / Al-Jadriya.Afterward, these samples were placed in the refrigerator at a temperature of 4 °C, after which the isolation and identification process was carried out.

Isolation and Identification
Infected samples of the fruiting bodies and the culture medium were washed separately with water for 10 minutes, then cut into small pieces (0.5-1) cm long, and sterilized externally using sodium hypochlorite solution (3% free chlorine) for 3 minutes.Subsequently, they were washed with sterile distilled water, dried well on filter paper, and planted in 9-cm Petri dishes containing 15-20 ml of Potato Dextrose Agar (PDA) , at a rate of 3 replication for each sample, four pieces in each plate, and incubated in the incubator for 3 days at a temperature (1 ± 25 °C).At that point, it was purified by taking a small piece from the edge of the fungal colony using a sterile needle and placing it in the center of another Petri dish containing the (PDA) medium and incubated in the incubator for 5 days.The growth of the fungal colony was observed until the plate was filled with the fungus Trichoderma spp.depending on the shape and color of the colony and the shape of the conidiophores.Besides, the spores formed using the taxonomic keys approved by / Plant Protection Department / College of the Agricultural Engineering Sciences / University of Baghdad.The frequency of pathogenic and associated fungi was calculated according to the equation of [27][28][29].
Frequency %=(Number of pieces in which mushrooms appeared on the plates) / (Total number of pieces used) × 100

Pathogenicity Test of Pathogenic Fungi
Healthy fruiting bodies belonging to the white mushroom (Agaricus bisporus) were brought from the local markets, washed and sterilized with sodium hypochlorite solution at a concentration of 3% for 3 minutes, and washed with sterile water.Then, the fruits were dried with blotting paper, and the fruiting bodies were cut into two halves and placed inside Petri dishes.A disk of 0.5 mm in diameter was taken from each of the two isolates of the fungus Trichoderma spp. it was placed on the fruiting body in two locations on the cap and on the stem, using a borer.So, the plates were covered with Parafilm, where 6 replicates were used for each isolate with 6 repetitions for the comparison treatment, and it was divided into two parts and placed inside the incubator at a temperature of (1 ± 25 °C).The other was placed in the refrigerator at a temperature of (1 ± 4 °C) for 5 days with continuous monitoring and according to the method of [31].After which the readings were taken and the disease severity with the fruiting body was measured according to the pathological evidence, 0 = without symptoms, 1 = 1 -10%, 2 = 11 -25%, 3 = 26 -50%, 4 = 51 -75%, 5 = < 75% using the equation of [30].

Molecular Identification of the Pathogenic Fungus Trichoderma Aggressivum Using the Polymerase Chain Reaction (PCR) Technique
After the initial identification of the pathogenic fungus T. aggressivum, the identification was confirmed using the polymerase chain reaction (PCR) method.The isolate that gave the highest pathogenicity was identified in vitro [31].The isolates of T. aggressivum were active on Potato Dextrose Agar (PDA) by single spore method and incubated in an incubator at a temperature of 2 ± 25 °C for seven days, and after the completion of the growth of the fungal colony of T. aggressivum, they were sent to Jisr Al Musayyib Company in order to carry out the extraction stages.

DNA Extraction from T.aggressivum
The DNA extraction process was carried out using Plant Kit, a commercial kit prepared by the Korean company Bioneer, following the approved steps of the company.

Replication of Extracted DNA
5 µl of extracted DNA, 1 µl of ITS1 primer, and 1 µl of ITS4 primer (Table 1) were added with 11 µl of ionic distilled water and placed in 0.2 ml small tubes containing 5 µl of Master Mix.The materials were mixed utilizing Expense and centrifuged for 15 seconds with shaking for 5 seconds, then the tubes were transferred to a thermal cyclers (PCR) device under optimal conditions for cycles Table (2) [32].The DNA ladder that was replicated was sent to the South Korean company (Macrogen Inc) for nucleotide sequence determination.

Gel Electrophoresis
Electrophoresis technology was adopted using a 1% agarose gel to detect DNA bands.The agarose gel was prepared by dissolving 1 g of agarose in 90 ml of distilled and sterilized water and 10 ml of a solution (10X TBE buffer).The mixture was heated in a microwave oven for 3 minutes, then the mixture was cooled and 5 µl of ethidium bromide was added to it.The mixture was mixed with light shaking, and then the agarose gel mold was prepared.The ultraviolet sterilized comb was placed at one end of a mold to make holes in the gel.The agarose gel was poured and left to cool and harden for 30-45 minutes at laboratory temperature.After that, the comb was removed and 5 µl of DNA ladder was added to the first hole (containing standard DNA).The same amount of extracted nucleic acid was added to the other holes, the device was covered with its cover, and electrophoresis was carried out at 70 volts for 60 minutes in order to detect the extracted and amplified DNA bands, which represent the PCR products.The DNA ladder was purified and the samples were sent to the Korean company (Bioneer) in order to determine the sequence of nitrogenous bases.

Sampling
The results sampling of 32 samples that were carried out from Agaricus bisporus cultivation sites, which were taken from the affected fruits, the casing, and the medium .Indeed, 33 isolates were isolated from Al-Wadaq farm (Baghdad governorate), 30 isolates from the Diwaniyah province site, 27 isolates from the Babylon province site, and 2 isolates from the Sulaymaniyah province site.All isolates showed many pathogenic and competitive fungi for the cultivation of the white mushroom A. bisporus such as Aspergillus spp., Cladobotryum spp., Penicillium spp., Phoma sp., Rhizopus spp., Trichoderma spp.and Ulocladium sp.This is consistent with many previous studies that showed the association of these fungi to the culture media of A. bisporus [33][34][35].

Isolation and Identification
The results of Table ( The fungus Trichoderma aggressivum is characterized by the formation of white colonies at the beginning of its growth on the culture medium (PDA), and it gradually turns green, starting from the center of the colony, and after five days it turns into a dark green color.The ideal temperature for its growth is between 25-30 °C, and it forms conidia smooth, oval, and green in color, formed on a cylindrical (phialide) borne on conidiophores, confined between the branches of the conidiophores, which are adjacent or not [15], [25], [26].Figure ( 1) confirmed a colony of the fungus T. aggressivum after growth on a PDA medium for seven days and microscopic characteristics using a 40magnification lens.

Pathogenicity Test of the Pathogenic Fungus Trichoderma Aggressivum on the Fruits of the White Mushroom Agaricus Bisporus in Vitro
The results in Table (4) and Figure (2) showed that all the fungal isolates tested at a temperature of 25 °C and 4 °C were effective in their pathogenicity on the fruiting bodies of the white fungus A. bisporus, which turned light brown five days after the fruiting bodies were inoculated with it.All Trichoderma spp.isolates at a temperature of 25 °C were recorded with a disease severity reached 60 and 60% for the two tested isolates, compared to the control treatment, which gave a disease severity of 10%.However, the above-mentioned isolates showed the least disease severity of the fruiting bodies of the white mushroom A. bisporus at a temperature of 4 °C, which recorded 40 and 50% for Trichoderma spp.compared to the control treatment, which recorded a disease severity of 0%.These results are consistent with previous studies, which showed that the pathogens that affect the fruiting bodies have a high pathogenicity that works to absorb nutrients from them, causing them to be small in size, change color, low weight, and total damage [31].In addition to the fact that these pathogens secrete enzymes such as cellulases, chitinases, and glucanase, as well as hydrolytic enzymes [37][38][39].
The reason that the control treatment showed a disease severity of 10% at a temperature of 25 ° C may be due to the sensitivity of the fruiting bodies to high temperatures for several days, which in turn leads to an increase in the speed of respiration.In addition to an increase in fruit ripening, which exposes them to infection with microorganisms that accelerate the process of deterioration and damage of the fruits.While the survival of healthy fruits at a temperature of 4 °C for several days prevented them from infection and damage, and this is considered one of the ways to increase the shelf life of fruits [40].

Results of the Gel Electrophoresis Phase
The results showed that the total DNA of the pathogenic fungus T. aggressivum under study was extracted, as the results of electrophoresis on agarose gel showed the presence of a band (one band) of the pathogen fungus with a molecular weight of approximately 600 bp for T. aggressivum, which was identical to the morphological identification of it (Fig. 3).

Reading of DNA Nucleotide Sequences of the Trichoderma Aggressivum
In comparison with the GenBank, The result of the nucleotide sequence of the fungus T. aggressivum obtained by Dr .Nawras by replication of the ITS region of the fungus using the general primer ITS4/ITS1 showed that there was a high similarity with the pathogenic fungus T. aggressivum.It amounted to 99% with the global isolates in the International GenBank National Center for Biotechnology Information (NCBI).The nucleotide sequence of the Iraqi isolates under study was deposited in the Gen Bank under the accession number (Table 5).As the identification of species T. aggressivum was the first time in Iraq on the fruiting bodies and media of the white mushroom A. bisporus.Table 5.The accession number of the Iraqi isolate to the fungus T.aggressivum.

Seq. Type
Accession number 1 aggressivum 5T OQ109172 It was found through the genetic affinity tree drawn by the SDTv1 program of the ITS region of the fungus T. aggressivum isolated from the fruiting bodies and the culture medium of the A. bisporus in Iraq with its equivalent counterparts from the gene bank, which recorded the highest similarity of 99% that the Iraqi isolate was lined up within one group with the global isolates, and the fungus, Rhizoctonia solani, was considered as a fungus outside the group, as in Figure (4).The Iraqi isolate of the T. aggressivum a similarity of 99% with its counterpart from the isolates (Turkey MH185822), South Africa (KX379163), Iran (MZ778797) and Czech Republic (FN549908). ) Depending on the molecular identification and sequence of the nitrogenous bases of the Iraqi isolate, it is possible to adopt the classification of the T. aggressivum isolated from the fruiting bodies and the culture medium of the white mushroom A. bisporus, which showed symptoms of infection with the pathogenic fungus T. aggressivum.The phylogenetic tree of Neighbor Joining was built from the nucleotide sequence of the ITS region of the T. aggressivum isolate that isolated from the fruiting bodies and culture media of the A. bisporus from the provinces of Baghdad, Babylon and Diwaniyah, respectively with their equivalents retrieved from the GenBank.The nucleotide sequence of R. solani was included for the purpose of comparison.Nucleotide analyzes were carried out using the MEGA11 program [40,41].

Figure 1 .
Figure 1.(A) T. aggressivum colony on culture media (PDA), (B and C) microscopic characteristics of T. aggressivum representing fungal isolation and reproductive structures.

6 Table 4 .•
Pathogenicity test of some isolates of the pathogenic fungus Trichoderma spp.for the fruits of the white fungus A. bisporus.Each number represents the average of six replicates.

Figure 2 .
Figure 2. Pathogenicity test of Trichoderma spp.isolates of the fruiting bodies of A. bisporus.A at 25°C, B at 4°C.

Figure 3 .
Figure 3. Electrophoresis of replicated DNA ladder from the genome of T. aggressivum (A = T. aggressivum) with a molecular weight of 600 bp, respectively, of the ITS region.standard DNA Ladder 100 base pairs.

Figure 4 .
Figure 4.The genetic relationships of the T. aggressivum.

Table 1 .
Primers used in the Polymerase Chain Reaction.

Table 2 .
Thermal polymerase program to amplify DNA extracted from T.aggressivum.

Table 3 .
3) showed the association of seven species of fungi associated with the fruits and culture media of the white mushroom Agaricus bisporus represented by the two pathogenic fungi Cladobotryum spp.and Trichoderma spp.Coupled with, the competitive fungi of A. bisporus, such as Aspergillus spp.and Penicillium spp.and Phoma sp. and Rhizopus spp., and Ulocladium sp.The most common pathogenic fungus was Cladobotryum spp. with a frequency of 56.2%, followed by Trichoderma spp., with a frequency of 54.1%, appeared in samples from two sampling areas.The species T aggressivum recorded a frequency of 42.5%, which was isolated and identified for the first time from the fruiting bodies and media of the A. bisporus.Furthermore, Aspergillus spp showed the existence percentage reached 65.5%, which is the highest among the competing fungi, followed by Penicillium spp., with an existence percentage of 26.6%, and then Rhizopus spp.,Phoma sp. and Ulocladium sp. with a frequency of 16.6%, 10.4%, and 10.4%, respectively.Fungi associated with the fruiting bodies and culture media of the A.bisporus.