Molecular study of pathogenic and saprophytic fungal species on infected parts of Malus pumila L. of district Qilla Abdullah, Balochistan, Pakistan

Apple (Malus pumila L) of the family Rosaceae, most cultivated fruit in temperate regions of the world and is used fresh or processed. The apple production is affected by several pathogens including fungi. The present study was designed to identify disease-causing agents that reduce fruit production in the district Qilla Abdullah of Balochistan, Pakistan, which is the main apple production area of the province. Three varieties of apple: ‘Tur-Kulu’ (‘Red Delicious’), ‘Shin-Kulu’ (‘Golden Delicious’), and ‘Kaja’ were selected. Infected leaf samples were collected from eight different sites of tehsil Gulistan, district Qilla Abdullah. The cultures of fungal micro-flora were grown on two media, potato dextrose agar (PDA), and malt extract agar (MEA) followed by incubation for one week. The resulting colonies were observed under a microscope and identified based on morphological characters. Predominant fungal species was identified through ITS marker and PCR amplification. The isolated pathogens belonged to Zygomycota and Ascomycota divisions. The pathogens found were Aspergillus niger, A. oryzae, A. terrus, Colletotrichum gloeosporioides, Fusarium oxysporum, Mucor spp., Penicillium expansum, and one species of Absidia as well as Rhizopus. Colletotrichum gloeosporioides were predominantly found in all varieties. Morphological and phylogenetic analysis confirmed the identity of AcademicPres Notulae Botanicae Horti Cluj-Napoca Agrobotanici Hasnain A et al. (2023). Not Bot Horti Agrobo 51(2):13167 2 Colletotrichum gloeosporioides. As a result of this study, the predominant pathogen species Colletotrichum gloeosporioides is one of the causes of leaf infection in apple varieties.


Introduction Introduction Introduction Introduction
Fruits are the most important food and source of essential vitamins for human beings (Sanzani et al., 2016). Apple (Malus domestica Borkh.) is one of the important ancient planted fruit crops in the world (Cornille et al., 2014;Duan et al., 2017). Initially it was found as wild fruit in central Asia and then spread all over Europe in pre-historical times. Apple's nutritional values make it unique from other fruits. Due to their excessive dietary values, the demand of apples is increased all over the world. It is the 3 rd most cultivated fruit in the world with an estimated production of 86 million metric tons (FAO, 2023).
Balochistan is a province of Pakistan and has the largest area among all provinces. It is also called "The Fruit Basket of Pakistan" due to the production of fruits. Balochistan provides 90% of fruits all over the country, with 34% production of apples (Samad, 2023). Apples are the fourth most important deciduous fruit grown widely in Balochistan. 'Shin-Kulu' ('Golden Delicious') and 'Tur-Kulu' ('Red Delicious') are famous varieties of apples in Balochistan due to their attractive colour and taste (Noonari et al., 2015). The most suitable climatic conditions and soil for apple growth prevail in the hilly areas of north Balochistan and KP province. The total area of Balochistan which is under cultivation is about 119.8 thousand hectares with an annual production of 427.9 thousand tons of apples (Khan et al., 2019). Most apples are grown in Qilla Saifullah, Quetta, Kalat, Qilla Abdullah, Ziarat, Zhob, Pishin, and some other districts of Balochistan (Nisar et al., 2011).
Due to its high nutritional value, apple is widely grown in the world. Its large cultivation in this area makes apples a host reservoir for many microorganisms (Abdelfattah et al., 2016;Liu et al., 2018). The production of apples is affected by different threats from pathogens and their toxins, such as mycotoxins (Fisher et al., 2012). Powdery mildew (Podosphaera leucotricha), bitter rot (Colletotrichum acutatum complex, Colletotrichum gloeosporioides complex), apple scab (Venturia inaequalis), black rot (Botryosphaeria obtuseco), and white rot (Botyrosphaeria dothidea), cedar-quince rust (Gymnosporangium juniperi-virginianae, Gymnosporangium clavipes, Gymnosporangium globosum), and sooty blotch/flyspeck (Geastrumia polystigmatis, Zygophiala jamaicensis) are fungal diseases with their causative agents that cause the most serious damage in apples (Holb, 2009;Nicole, 2019). Previous studies indicated that fungi caused premature fruit drops that become the source of 50% losses in the yield of apples (Raja et al., 2017). Alternaria alternata produce mycotoxins which are responsible for the core rot and mouldy core of apples in some regions of the world (El-Mohamedy and El-Sayed, 2015).
Several fungi, including Alternaria alternata, Cladosporium cladosporioides, Fusarium semitectum, and Penicillium spp., produce mycotoxins which were responsible for apple fruit drops and also harmful to human beings. But amongst all pathogens, Alternaria alternata is responsible for fruit drops in apples (Youssef and Roberto, 2020). The Alternaria spores could be carried into the apple cavity by mites (Van der Walt et al., 2011). Fungi cause the most severe diseases in plants. When the fruit is growing on a plant or stored, the fungi grow on it and produce mycotoxins that are dangerous for consumption by human beings. The Monilinia and Penicillium species are also reported to cause spoilage of fruit in apples (Alwakeel, 2013).
The aim of this study was identification of predominant fungal pathogens based on their morphological and molecular characteristics that affect the growth and yield of apples in the district Qilla Abdullah of Balochistan province of Pakistan.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Study area and sample collection Tehsil Gulistan, district Qilla Abdullah, Balochistan was selected for the research purpose. The main reason for selecting the site was its richness in apple production. The diseased plant parts of various apple varieties were collected from eight different sites. These sites were Adrhamanzai, Batayzai, Habibzai, Imranzai, Kolazai, Masiyzai, Mayzai, and Slumankhail. Three varieties of apple: 'Tur-Kulu' ('Red Delicious'), 'Shin-Kulu' ('Golden Delicious'), and 'Kaja' (Figure 1) were selected based on infection prevalence on leaves, branches, and fruits.

Identification of disease
The infected leaves, branches, and fruits from apple trees were collected in zipper bags. Two methods i.e., direct identification and culturing methods were applied to investigate causative agents of infection (Skyes and Rankin, 2014).

Direct identification
Some diseases were directly identified by assessing the physical condition of diseased plant parts. The infection was also studied using a microscope. The spores and mycelia from infected parts of plants were placed on a slide containing a trypan blue stain. The materials were spread on the slide, a cover slip was placed and the slide was observed under a microscope.

Culturing method
Preparation of media The choice of culture medium depends on the type of microorganism to be isolated for identification purposes.
Fungal culture media The most direct and often the most convincing way to establish a diagnosis of a fungal infection is to culture the fungus from an infectious sample. Two types of fungal media, malt extract agar (MEA) and potato dextrose agar (PDA) were used to culture fungus (Black, 2020). Saline suspension of specific spores from diseased plant parts was collected and spread on the media plates under sterile conditions. Agar media plates 4 and MEA, and PDA plates were incubated at 37 °C and 28 °C, respectively for a week. The fungal colonies were observed and the pure cultures were maintained.
Identification of fungal pathogens For slide preparation, different colonies from culture plates were picked using a sterilized inoculating loop, macerated finely onto slides containing a drop of trypan blue, and then covered with coverslips. Slides were observed using a light microscope at different magnifications (4X, 10X, and 40X). Fungal species were identified on basis of their morphological characters (Shamly et al., 2014).
Molecular identification of predominant pathogen fungi DNA extraction and quantification Molecular identification of predominant pathogens was done by taking fungus samples from the infected part of all varieties of plants through isolation of Genomic DNA, by using the CTAB method as described by Gardes and Bruns (1993) with some modifications the extracted DNA was visualized by agarose gel electrophoresis (Lee et al., 2012) and quantified on a double beam spectrophotometer Morris (2015).
Amplification and sequencing of fungal ITS region For the amplification of the fungal Internal Transcribed Spacer (ITS) region, ITS-1 (forward primer) GCTGCGTTCTTCATCGATGC and ITS-4 (reverse primer) TCCTCCGCTTATTGATATGC were used (White et al., 1990). The total volume of the PCR reaction mixture was 20 µL comprising PCR Master Mix: 12 μL, DNA template: 1 μL, forward Primer: 0.5 μL, reverse primers: 0.5 μL, and dd. water 6 μL. The Amplified product of PCR was examined by 1% of agarose gel electrophoresis. The PCR product was sequenced by Capillary Electrophoresis Sequencing on ABI 3730xl System.

Sequence alignment and phylogenetic analysis
The obtained sequences were read, aligned, and edited to get a consensus sequence on BioEdit (https://bioedit.software.informer.com/7.2/). The new sequence generated in this study was compared with the closely related sequences of other Colletotrichum species, retrieved from GenBank. All the sequences of the ITS region were aligned using MUSCLE alignment software which generated a final dataset of 571 positions. For phylogenetic analyses, the tree was constructed based on Maximum Likelihood (ML) method. The Tamura-Nei model was chosen with the lowest BIC scores (Bayesian Information Criterion) (Tamura and Nei, 1993). Maximum likelihood analyses were performed with MEGA11 with 100 replicates (Tamura et al., 2021). Less than 50% of bootstrap support on the nodes was considered insignificant and was not shown.

Direct identification of samples
The direct identification method identified five types of disease symptoms in the leaves of apple trees.

Identification of fungi responsible for apple diseases
The fungi isolated from leaves and branches were identified from infectious sites based on colony morphology and microscopy. Colony features (color, shape, size, and hyphae) were observed under a stereomicroscope. Microscopic characters such as conidia, spore, hyphae, and other structures were observed using a lactophenol cotton-blue stained slide mounted with a small portion of the spore and mycelium (Gaddeyya et al., 2012). The identification keys of Frisvad and Samson (2004) were used to classify isolates belonging to different genera.

Variety 1. 'Shin-Kulu' ('Golden Delicious') Aspergillus niger
Colonies were black having light brown conidia, round with spherical to globose vesicles, rough texture, irregular margins, 40-50 mm in one week; one conidial head on the tip of aseptate conidiophores; conidial head bearing spores were observed; conidia were dark black and globose as shown in the Figure 3 (a).
Mucor sp. 1 Colonies were cottony to fluffy in appearance, white to yellow; erect sporangiophore and the spores were present in the sporangium, colour of sporangium was dark green at the top, with light green at the center as shown in Figure 3 (b).

Colletotrichum gloeosporioides
Colonies were gray to black, regular, cottony, and raised, smooth margins, white aerial mycelium covering the whole surface of the plate, hyaline conidiophore, conidia cylindrical with a round end, hyphae hyaline, as shown in Figure 3 (c).

Fusarium verticilliodes
Colonies were white to creamy and pink, rough texture, irregular margins; conidia were elongated and kidney shaped in sporodochium as shown in Figure 3 (d). Figure 3. Figure 3. Figure 3. Colonies were white to greyish black brown, rough texture, uneven, irregular, filamentous cottony and fluffy growth; well-developed rhizoid; hyaline to globose, light brown sporangium, round and terminal as shown in Figure 4 (a).

Penicillium expansum
Colonies white and greyish; bearing conidiophores; having brush-like conidia as shown in Figure 4 (b).
Absidia sp. Colonies were cottony white, hyphae were septate and long; sporangiophores were alone and branched; columella was present; conidia were spherical and long in structure as shown in Figure 4 (c).

Aspergillus oryzae
Colonies were greyish brown; rough texture; circular and irregular margins; vesicles possessed stigmata bearing conidia; conidia were yellow with septate conidiophores; hyaline; thick-walled hyphae as shown in Figure 4 (d).

Fusarium oxysporum
Colonies hyphae were septate and non-pigmented (with trypan blue stain). There was an aspherical chlamydospore as shown in Figure 5 (b).

Aspergillus terreus
Colonies were black-brown, hair-like soft tufts and stipe, coarsely roughened texture, irregular, granular, hard, sticky margins, thick and double-walled hyaline conidiophore as shown in Figure 5 (d).

Colletotrichum gloeosporioides
Colonies were Greyish to white, regular, smooth borders, hyaline conidiophore, conidia round ended; as shown in Figure 5  The apple trees of Qilla Abdullah were infected by different infections. Fungal infection symptoms were caused by members of Ascomycota, and Zygomycota, and were identified in diseased apple parts in the current study. Various fungal species such as Absidia sp., Aspergillus oryzae, A. niger, A. terrus, A. glaucus, Colletotrichum gleosporoides, Fusarium verticilloides, Fusarium oxysporum, Penicillium expansum, Mucor sp.1, Mucor sp. 2, and Rhizopus microsporus were isolated and identified from diseases apple trees as given in Table 1

Molecular identification
Genomic DNA extraction and quantification DNA from the predominant pathogenic fungus was extracted from samples obtained from three apple varieties (T1, T2, and T3) by the CTAB method with some modifications. DNA quantification was done by using UV/VIS spectroscopy at the absorbance of 260 and 280 nm respectively.
Amplification of ITS region ITS primers were used for PCR amplification the size of the PCR product was 560bp which indicated that ITS regions were amplified. The result of the gel is shown in Figure 6.  Figure 6. Figure 6. Figure 6. The result of ITS markers on genomic DNA of Colletotrichum spp Molecular identification by BLAST The cleaned and edited sequence of fungal ITS region was BLAST searched on the NCBI website for closely related sequence homology. The query sequence was matched with Colletotrichum sp., indicating that the predominant pathogen infecting all three apple verities was C. gloeosporioides. Figure 7 shows a rectangle cladogram of the neighbour joining distance tree of results obtained from BLAST in which nearest clade of unknown sequence comprised of C. gloeosporioides.  Phylogenetic analysis This study generated 1 new sequence which has been submitted to GenBank (OR091360). The ITS dataset included 32 taxa and 571 sites, that comprised of 410 conserved, 157 variable, and 77 Parsimony informative sites. Phylogram included 31 in group taxa (all belong to genus Colletotrichum) and 1 out group taxon (Monilochaetes infuscans, NR155365). Our sequence of Colletotrichum gloeosporioides is clustered with the other collection of the same species (MN170560) in a well resolved clade (Figure 8) therefore, confirms its identity. The apple is one of the most important fruit species, cultivated in the temperate regions of many countries, which has fruits with a pleasant and aromatic taste, rich in nutrients. Apples are an important source of vitamins, minerals, and carbohydrates, having an important nutritional value and being beneficial for human health (Patocka et al., 2020;Fotirić Akšić et al., 2022;Sestras and Sestras, 2023). Pakistan produced 589,171 tons annually and ranked 25 in apple-producing countries, while China is the largest apple producer with 40,393,000 tons of production. As apple trees are susceptible to different diseases which decreases the yield and quality of apples. In northern Balochistan, about 60% of the fruit trees have dried and the remaining is unlikely to survive due to the declining water and diseases. In recent years, in Quetta, Qilla Abdulah, Loralai, Qilla Saifullah, and Pishint, the yield of apples is affected by different diseases. It is estimated that the production of apples has reduced by at least 50% due to many reasons such as the impact of climate change, the shortage of water, fungal diseases, and pest attacks. Fungal diseases can cause yield losses in apple orchards and affect fruit quality (Siddique et al., 2007).
Penicillium expansum affects the bulbs and fruits of apple plants. Symptoms appear as oval, soft yellowbrown watery spots on the fruits (Sandor, 2008). P. expansum is one of the main pathogens causing the decay of fruits and vegetables. Postharvest fungal diseases of apples are mainly caused by P. expansum and Rhizopus sp. Both caused soft rot, a postharvest disease in apple trees (Kwon et al., 2011). Fusarium sp. has caused wilt disease in apples resulting in yellow leaves with interveinal chlorosis. Hence leaves fall off thus causing wilting of apple leaves in Qilla Abdullah, Balochistan.
In the present study, the PCR assay was used to check the predominant fungus infecting all varieties of apples by amplifying and sequencing the ITS region. The phylogenetic analysis in the present studies find out that the pre-dominant fungi Colletotrichum gloeosporioides are found to infect all the selected varieties of apples. This research result was matched with the study in which C. gloeosporioides were detected with other Colletotrichum species like C. godetiae and C. acutatum causing bitter rot disease (Sanzani et al., 2012) infected the mangoes. Another study revealed the same result in which seven different Colletotrichum species including C. gloeosporioides species complex isolated from three different apple orchards, causing bitter rot in apples and strawberries in Belgium (Grammen et al., 2019).
Overall, isolated pathogens can cause hazardous diseases which reduce not only the apple yield but also affect the growth of apple trees. Among identified fungi, such as P. expansum and Rhizopus sp. also caused postharvest diseases of apples further aggravating the attack of fungal diseases C. gloeosporioides.

Conclusions Conclusions Conclusions Conclusions
The present research specified the apple varieties such as 'Golden Delicious' ('Tur-Kulu') and 'Red Delicious' ('Shin-Kulu') which are more susceptible to fungal diseases. There is a lack of research work on the identification and isolation of apple diseases. The current study isolated and identified fungal pathogens and associated fungi for the first time from the apple trees of Qilla Abdullah. The total abundance of isolated genera was 11, belonging to the division Ascomycota and Zygomycota. The symptoms of wilting were present in plants growing all over the district and the infected leaves showed purplish-yellow spots. The nine pathogenic fungi from apple trees suggest the presence of several other fungal pathogens that needs to be investigated. The molecular analysis showed that C. gloeosporioides is pre-dominant in all collected varieties and the most prominent fungal pathogen causing bitter rot disease in apples. Identification of the disease-causing agents and precautions before the spread of the disease is very essential, which helps in the diagnosis and early detection of the pathogen. It may be helpful to maintain check and balance in the planting material for the prevalence of any potential pathogen even before the crop is sown. This fungal taxon can be very detrimental to apple yields, 13 so preventive measures must be taken to reduce the possibility of the causative disease. Adequate antifungal sprays and awareness are warranted in all districts and provinces to ensure an increase in total apple fruit production.
Authors' Contributions Authors' Contributions Authors' Contributions Authors' Contributions MMK and AH: collected samples and experimental work; H-HY, ZI: complied all data; NY, TA and AT: morphological and molecular identification; SAZM: final editing; AE, B, H, MI, HNA: DNA isolation and PCR; AJ: English language and grammar checking.
All authors read and approved the final manuscript.
Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.