Diversity of endophytes associated with the medicinal plant Pulicaria crispa

: Pulicaria crispa is a medicinal and aromatic plant that is distributed around the world. In this study, a remarkable variety of endophytic fungi and bacteria were isolated from different parts of P. crispa . Fourteen endophytic fungal species belonging to seven genera ( Cladosporium, Chaetomium, Volutella, Emericella, Aspergillus, Penicillium, and Ulocladium ) were recorded . The relative abundance of genus Aspergillus was 40.5% (recorded the highest count of genera). Volutella ciliata registered the highest fungal species count with a relative abundant of 16.2% and it is the first record of this fungus in Aswan region. Five bacterial species belonging to the genus Bacillus including Bacillus sianensis , B. safensis , B. subtilis , B. altitudinis , and B. vallismortis were isolated . B. sianensis which colonized roots represented the highest relative abundance percent (32 %). This is the first study of the microbes' diversity inside the tissue of the medicinal plant P. crispa .

These creatures inhabit undamaged tissues of the host plant and perform a wide range of biological interactions ranging from symbiotic to slightly pathogenic (Strobel et al., 2005).The interaction between the plant and endophytes is a symbiotic relationship based on the benefit of both the plant and the microorganism (Polesi, 2011;Hardoim et al., 2008).As a result of this wonderful relationship, the microbes inside the plant produce a group of substances (secondary metabolites) with unique structures that cannot be obtained from a chemical reaction and which are of great importance to the plant (Carroll, 1988;Schulz et al., 1998;Parker, 1999;Redman et al., 2001;Schulz et al., 2002;Strobel and Daisy, 2003;Lee et al., 2004;Dudeja et al., 2012).
Fungi and bacteria that share the same environment (i.e., the same food sources and the same dwelling) compete for resources and a number of relationships appear, including antagonism.This relationship organizes and protects a community in which microbes live, which leads to supporting the stability of this assemblage by limiting the excessive growth of these endophytes and by fighting foreign microorganisms that want to harm them and the environment in which they live (Bharati et al.,1982;Mille-Lindblom et al., 2006;Zettler et al., 2013;Wang et al., 2015;Kumar et al., 2015;Feichtmayer et al., 2017;García-Bayona and Comstock, 2018;Karunasinghe et al., 2020;Zhang et al., 2021).This study aims to screen the diversity of endophytic bacteria and fungi in the medicinal plant Pulicaria crispa.

Isolation and identification of endophytic fungi and bacteria
The plant segments were placed on the surface of potato dextrose agar (PDA) plates supplemented with chloramphenicol (0.5 g/L) for fungal endophytes and nutrient agar (NA) plates for bacterial endophytes.PDA plates were incubated at 28 °C for 3 weeks, while NA plates were incubated at 37 °C for 72 h.The growth was daily observed (El-Zayat et al., 2008).
Fungi were identified based on their morphological and microscopic characteristics, including the color and texture of the colony as well as the color and shape of the conidia and hyphae (Moubasher (1993).The bacterial isolates were molecularly identified by 16S rRNA gene sequencing using the universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-TCC TCC GCT TAT TGC TAT GC-3') (Frank et al., 2008).The obtained sequences were aligned with those deposited in the NCBI database to detect the similarity percentages using the Blast tool from the website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).The sequences of the present isolates were submitted to the NCBI gene bank to gain accession numbers.A neighborjoining phylogenetic tree was constructed by MEGA X software (Kumar et al., 2018).

Results and discussion
In the present study, fungi and bacteria inhabiting the medicinal plant P. crispa were screened.According to our knowledge, this is the first study to report the endophytic fungi and bacteria in P. crispa.
Fourteen endophytic fungal species belonging to 7 genera were isolated from different plant organs, including roots, stems, leaves, and flowers (Table 1).The roots had the highest fungal colonization where they recorded 15 colonies/segment, while the fungal colonization in the other organs of the plant, including the leaves, stems, and flowers, recorded 9, 8, and 5 colonies/segment, respectively (Table 1 & Fig. 1).
In the whole plant, the genus Aspergillus recorded the highest count of genera (15 colonies/segment and relative abundance 40.5%), followed by Emericella with colonization counts 7.0 colonies/segment and relative abundance 18.9%.Volutella ciliata recorded 6.0 colonies/segment and relative abundance 16.2%.This fungus represents the first record in Aswan region despite isolation of it from various parts of the world.Volutella ciliata protect plants against potentially toxic compounds in the soil Vaughan et al., 1993) and has a role in recycling and accumulation of organic matter in soil (Osono and Takeda, 2007).Cladosporium and Chaetomium count was 4.0 colonies/segment with relative abundance 10.8% and 3.0 colonies/segment with relative abundance 8.1 % respectively.The lowest colonization was recorded with both Penicillium and Ulocladium (only one colony/segment and relative abundance 2.7% for each) (Table 1 & Fig. 2).
The highest count of fungal species was V. ciliata (6 colonies/segment) with a relative abundant of 16.2%.The relative abundance of A. niger, A. terreus, C. cladosporioides and E. quadrilineata was 10.8%, and the total count of 4 colonies/segment while the remaining fungal species relative abundance was ranged between 2.7%-5.4%,and colonies count between 1-2 colonies/segment (Table , 1 & Fig. 3).
The second rate of isolation was recorded in flower at 18.75% by isolation of V. ciliata followed by leaf at 14.29% for two genera Aspergillus and Cladosporium.The first genus was represented by A. flavus, A. fumigatus, A. sydowii, and A. terreus (3.57% each).Cladosporium is represented by only one species C. cladosporioides and isolated only from the leaf.Also A. sydowii inhibited only leaves in this plant (Table 2 & Fig. 4).
Rate of endophytes isolation in the stem was in the next order 11.54% represented by the genus Chaetomium (C.cochliodes 7.7% and C. globosum 3.85%) which was also specific to stem, 7.70 % for E. nidulans and 7.69% for genus Aspergillus (A. flavus and A. fumigatus, 3.85% each).The rate of isolation (6.25%) was recorded in flowers for both A. niger and U. chartarum while 3.57% was noticed in leaves for E. nidulans.Ulocladium chartarum was isolated only from the flower and P. duclauxii from the stem (3.85%) (Table 2 & Fig. 4).
Endophytic fungi belonging to the genera Aspergillus and Penicillium were isolated from medicinal plants found in Saudi Arabia, including Pulicaria crispa, to know its antioxidant ability of the extracts extracted from these fungi.Cladosporium and Ulocladium were isolated from medicinal plants belonging to the family Asteraceae, as they produce the enzyme Lasparaginase, which is used in the treatment of cancer.Aspergillus and Penicillium were also isolated during the study conducted on medicinal plants belonging to Asteraceae and found in countries with biodiversity: China, Thailand and Iran (Hatamzadeh et al., 2020;Caruso et al., 2020;Hassane et al., 2022).The genera Cladosporium, Penicillium, Aspergillus and Chaetomium has been isolated from plants other than this plant and does not belong to Asteraceae such as Oryzae sativa L. and Ricinus communis linn.plants (Sandhu et al.,2014).Penicillium sp., and Aspergillus niger were isolated as endophytic fungi from medicinal plants in Nigeria, Alstonia boonei, Enantia chlorantha and Kigelia Africana (Adeyemi, 2015).Aspergillus terreus is obtained from the leaves of the Bacopa monnien plant (Soni et al., 2021).In addition endophytic fungi belonging to the genus Penicillium and Aspergillus were obtained from fruits of the Chaenomeles plant (Lykholat et al., 2021).Phoma, Cladosporium, Alternaria and Bipolaris were got from Caralluma acutangula, Rhazya stricta and Moringa peregrina plants.These microorganisms have been studied in order to know the types present in plants and their impacts on development of seeds and reducing oxidative processes (Khan et al., 2017).Five endophytic bacteria were isolated from P. crispa (Table 3).The results of sequences alignments revealed that the isolates sp1, sp2, sp3, sp4 and sp5 were highly similar with Bacillus safensis strain NBRC100820 (99.75%),Bacillus subtilis strain DSM 10 (99.04%),Bacillus altitudinis 41KF2b (100.00%), Bacillus vallismortis strain NBRC 101236 (99.93%) and Bacillus siamensis KCTC 13613 strain PD-A10 (97.05%), respectively.The 16S rRNA gene sequences of the present isolates sp1, sp2, sp3, sp4, and sp5 were deposited in the NCBI under the accession numbers OQ874962, OQ874964, OQ874811, OR731260, and OQ874990 (Fig. 5).
Many endophytic bacterial species were isolated from plants belonging to the genus Pulicaria, including Bacillus cereus, Agrobacterium farum, Bacillus substilis, Brevibacillus brevis, Acinetobacter rodioresistant, and Burkholderia cepacia (Fouda et al., 2021).Pulicaria crispa is a desert plant found in multiple countries, including Egypt, Sudan, Saudi Arabia, and the Sultanate of Oman.It is considered an aromatic medicinal plant with a strong smell and has been used in the medical field since ancient times.The plant's medical benefits are attributed to its important compounds such as flavonoids (phenolic compounds found in the plant that help prevent mutations, and aid in cancer treatment) (Panche et al., 2016), saponins (complex natural compounds in the plant with known for their anticancer properties and immune system enhancement) (Shi et al., 2004), tannins (phenolic compounds that act as antioxidants by strengthening oxidative stability) and alkaloids (compounds used to treat malaria and cancer due to their aesthetic properties) (Tong et al., 2022).
It was interesting to find that fourteen fungal species belonging to six genera and five bacterial species can be adapted to the antimicrobial compounds produced by this medicinal plant.Furthermore, these microbes can coexist with each other, fungi with fungi, bacteria with bacteria, or fungi with bacteria, without causing harm.However, further research is needed to understand the mechanisms behind their ability to live together.Consequently, our laboratory will be investigating the antagonistic reactions between these microorganisms.

Conclusion
This study screened the fungal and bacterial communities inhabiting the medicinal plant P. crispa.Endophytic microorganisms, including bacteria and fungi, were found to coexist within the plant.Despite the plant's production of many antimicrobial compounds, such as alkaloids, saponins, and terpenes, the isolated endophytes were able to reside within the plant tissue.Furthermore, these endophytes were able to coexist within the same tissue without causing harm to each other.Interestingly, certain bacteria and fungi were specific to particular parts of the plant.These findings have promoted further investigation into the mechanisms by which medical plant-associated microorganisms can coexist, with a focus on studying their antagonistic properties in further research.

Fig. 1 .
Fig. 1. variation of fungal total counts in different organs of P. crispa.

Fig. 4 .
Fig. 4. The percentage of isolation of endophytic fungal genera associated with P. crispa.

Fig. 5 .
Fig. 5.A neighbor-joining phylogenetic tree with 1000 bootstraps shows the relatedness between the isolated strains and closely related members of the genus Bacillus derived from NCBI GenBank using the MEGA X software.