Fungi were isolated from the outer and inner bark of Taxus chinensis var. maire in Baiji, Anhui Province, China. The endophytic fungus was aerobically cultured in potato dextrose broth medium at 28 ℃ for one week. Plackett-Burman design: In this study, seven factors were included. For each factor, a high (+) and low (-) level was tested. Twelve runs of various levels of factors were formulated by Design-Expert version 8.0 and the response was measured according to the yield of taxol. All runs were conducted in 250 mL Erlenmeyer flasks containing 100 mL PDA liquid medium at 30 ℃ with an initial pH of 7.0. The tested factors were: sodium benzoate; salicylic acid; phenylalanine; sodium acetate; glycine; CuSO₄; methyl jasmonate. After culturing for 8 days, the fungal taxol was extracted as described above.

Authenticated Axonopus leptostachyus was collected in the city of Pocone, Pantanal of Mato Grosso, Mato Grosso, Brazil, in April 2012. The endophytic fungus Aspergillus terreus P63 was isolated from roots of the grass Axonopus leptostachyus, which were subjected to surface sterilization. The endophytic fungus strain A. terreus P63 was cultivated in two 500 mL Erlenmeyer flasks, each containing 30 mL of distilled water and 25.0 g of rolled oats. The media were autoclaved two times (in two consecutive days) at 121 ℃ for 20 min. Following sterilization, approximately 4 small pieces (2 × 2 cm) of PDA medium, from the Petri dish containing biomass of the A. terreus P63 isolated was inoculated into each flask and the flasks were sealed with cotton to permit aerobic growth. The media inoculated with the endophyte were incubated while stationary at 25 ℃ for 30 days in the dark.

The plant, Huperzia serrata (Thunb.) Trev., was collected in Xichou County, Yunnan Province, China, in July 2013. Botryosphaeria sp. 483 was cultured on PDA at 28 °C for 16 days.

The fungal strain C. globosum was separated from the sterilized leaves of G. biloba, a medicinal plant growing in Linyi, Shandong province, China. The fungus C. globosum was cultivated on PDA medium for 5 days at 28 ℃ to provide the culture broth (30 L).

Isolation of endophytes: The leaves of 24 plant species were collected in the Amazon Rainforest biome, in Cayenne and Roura, French Guiana. After collection, the plant material was washed with sterile water and surface sterilised by sequential immersion in 70% aqueous ethanol (3 min), followed by 5% aqueous sodium hypochlorite (5 min) and finally by 70% aqueous ethanol (1 min). After these procedures, the leaves were rinsed with sterilised water. The residual sterilised water was incubated in Petri dishes to ensure that all the epiphytic microorganisms were eliminated. The surface-sterilised leaves were cut into 64 small pieces which were placed in 2 ml Eppendorf tubes containing 1 ml of potato dextrose agar medium (PDA, Fluka Analytical, Germany) at 26 ℃. Each individual hyphal tip of emerging fungi was removed and placed on a sterile PDA culture medium in 10 cm Petri dishes. The leaf fragments were cultured for a maximum of 5 months. All strains, including bacteria, were cultivated on solid PDA medium at 26 ℃ for 15 days, on 3 Petri dishes of 14 cm diameter (150 cm²). On the large scale, the microorganisms were cultivated under the identical conditions with 130 Petri dishes of 14 cm diameter (2 m²).

The culture of C. dematium used in this study was obtained as an endophyte from a small cutting taken from an immature Pteromischum sp. plant collected in a Caribbean costal Costa Rican rainforest. Isolate CR-12 (17 l) was grown in shake culture for 28 days at 25 ℃.

PR4 was isolated as an endophyte from the rhizome of Picrorhiza kurroa. Picrorhiza kurroa Royle ex. Benth (Plantaginaceae) is a perennial herb endemic to the north western alpine Himalayas. The endophyte PR4 was grown on PDA and in PDB at 26 ℃ for 15 days with constant shaking at 200 rpm in the latter case.

The two candidate NR-PKSs (PKS_3671 and PKS_4063) show differences in their domain organizations. PKS_3671 possesses two ACP-domains. Apart from that, only PKS_3671 contains a SAT-domain . These domains provide the first building block in the polyketide assembly, which usually is different from the extender unit malonyl-CoA (also known as the 'starter unit effect'). The ACA-synthesis however is believed to involve merely malonyl-CoA molecules. Even though the ACA-producing PKSs MdpG, ACAS, EncA, AptA and ClaG contain SAT-domains, an amino acid sequence alignment of these domains revealed that they all lack the active-site cysteine in the GXCXG motif and therefore most likely have no acyl transferase activity. Instead, all malonate building blocks are assumed to be loaded by the MAT. Under this aspect, the SAT-domain of PKS_3671 (that includes the correct GXCXG motif) likely incorporates a starter unit different from malonyl-CoA indicating that this enzyme is not involved in the biosynthesis of ACA. Therefore, the ACA-synthesizing PKS in C. asteris would rather be PKS_4063 that misses the SAT-domain .In the monodictyphenone and cladofulvin pathways, the cluster-encoded gene products MdpH and ClaH are crucial enzymes pushing the biosynthesis towards emodin. These EthD-domain-containing enzymes are suggested to catalyze the decarboxylation of ACA (3) into atrochrysone (4). Surprisingly, no such EthD-domain is encoded in the whole C. asteris genome. On the other hand, four genes directly attached to the putative ACA-synthase-coding gene pks_4063 show high similarity to genes of non-investigated PKS clusters in other fungi , which indicates an involvement in tailoring reactions of the respective polyketide pathways. According to InterProScan and BLASTp analyses, the genes sky_4060-62 encode a dehydratase and two dehydrogenases potentially catalyzing the multistep conversion of ACA (3) into emodin (1). Gene sky_4059 codes for a monooxygenase that putatively can connect two emodin molecules to the final product skyrin (2) in the style of the monooxygenase ClaM involved in the dimerization of the bisanthraquinone cladofulvin. Thus, the presence of these genes in the gene cluster gives further support to the hypothesis that PKS_4063 is the ACA-synthase in C. asteris. Mutational studies will be done in order to confirm these assumptions after a gene transfer system for this strain has been developed.

The seeds and roots of five adult healthy plants of P. cupana from each clonal cultivars CMU 871 (tolerant to anthracnose and oversprouting) and CMU 300 (tolerant to anthracnose and susceptible to oversprouting) were collected in November 2014 in the Embrapa (Brazilian Agricultural Research Company) experimental farms located in the cities of Manaus (2° 53′ 29.14″ S and 59° 58′ 39.90″ W, 99 m high) and Maues (3° 22′ 54″ S and 57° 42′ 55″ W, 18 m high), State of Amazonas, Brazil. The plant material was collected at the end of the dry season, when the accumulated rainfall and average temperature were respectively 196.0 mm and 28.46 ℃ in Manaus, and 272.3 mm and 28.17 ℃ in Maues. Five millimeter diameter plugs of each endophytic fungus were place at the center of Petri dishes (90 mm diameter), each containing 20 mL of PDA, and cultured for 13 days at 25 ℃.

Healthy cladodes and flowers of the invasive plant O. dillenii were collected from the Bundala National Park in the South-East arid zone of Sri Lanka on July 2013. Endophytic fungus that exhibited promising antimicrobial activity was cultured in 200 medium size Petri dishes (100 × 20 mm) of PDA for 17 days at room temperature.

The endophytic fungal strains were isolated from trees of the species A. dimidiata E. Mey. ex Arn (Icacinaceae) growing in the Western Ghats region, a mega-bio-diversity hot spot in India. The location coordinates of Periya, the collection locality are 11° 50′ 0″ North, 75° 50′ 0″ East. For each pure isolate, single hyphal tips were incubated in 250 ml conical flasks containing 50 ml of pre-sterilized PDA broth. Flasks were agitated at 200 rpm on a rotary shaker at 28℃ for 4 days.

The endophytic fungal strains were isolated from trees of the species A. dimidiata E. Mey. ex Arn (Icacinaceae) growing in the Western Ghats region, a mega-bio-diversity hot spot in India. The location coordinates of Periya, the collection locality are 11° 50′ 0″ North, 75° 50′ 0″ East. For each pure isolate, single hyphal tips were incubated in 250 ml conical flasks containing 50 ml of pre-sterilized PDA broth. Flasks were agitated at 200 rpm on a rotary shaker at 28℃ for 4 days.

Samples of C. acuminata were collected in April from the Botanical Garden, South of Guiyang, China. Small terminal, limbs (1x25 cm) from one to two years old were harvested and stored in a sealed plastic bag at room temperature and processed within 2 days. The fungi were statically cultured for 10 days at 28 ℃ in petri dishes. They were incubated into 1000 mL flasks containing 500 mL of PDA broth. After 4 days of incubation at 28 ℃ on rotary shaker at 150 rotations per minute (rpm), mycelia and broth were separated by filtration.

Isolation of fungal material: The fungus Fusarium solani was isolated from fresh healthy roots of Cassia alata Linn. The plant samples were collected from conservation forest of Gazipur, Bangladesh in August, 2013. Fusarium solani species (internal strain no. CARE-1), which had been isolated following surface sterilization from the root of the plant C. alata were cultivated at 28 ℃ for 28 days on potato dextrose agar medium.

Healthy samples (stems and roots) of Nerium indicum were collected from Heng Mountain, Hunan province, China, and immediately packed and sent back to our laboratory within 24 h. The endophytic fungi initially were cultured on PDA plates at 27 ℃ for 3 days. Then, the endophytic fungi were inoculated in 250 mL Erlenmeyer flasks with 100 mL fermentation medium containing under a constant temperature incubator shaker (ZHWY-2102C; Shanghai Zhicheng Analytical Co., Ltd, China) in order to produce secondary metabolites. The fungi were grown at 27 ℃ at 170 rpm for 3 days and then incubated under still conditions for 4 days.

Isolation of endophytes: The leaves of 24 plant species were collected in the Amazon Rainforest biome, in Cayenne and Roura, French Guiana. After collection, the plant material was washed with sterile water and surface sterilised by sequential immersion in 70% aqueous ethanol (3 min), followed by 5% aqueous sodium hypochlorite (5 min) and finally by 70% aqueous ethanol (1 min). After these procedures, the leaves were rinsed with sterilised water. The residual sterilised water was incubated in Petri dishes to ensure that all the epiphytic microorganisms were eliminated. The surface-sterilised leaves were cut into 64 small pieces which were placed in 2 ml Eppendorf tubes containing 1 ml of potato dextrose agar medium (PDA, Fluka Analytical, Germany) at 26 ℃. Each individual hyphal tip of emerging fungi was removed and placed on a sterile PDA culture medium in 10 cm Petri dishes. The leaf fragments were cultured for a maximum of 5 months. All strains, including bacteria, were cultivated on solid PDA medium at 26 ℃ for 15 days, on 3 Petri dishes of 14 cm diameter (150 cm²). On the large scale, the microorganisms were cultivated under the identical conditions with 130 Petri dishes of 14 cm diameter (2 m²).

A. terreus was isolated from the internal tissue of the healthy roots of Carthamus lanatus L. (Asteraceae), which collected from the wildly growing plant at Al-Azhar University campus in February 2013. Mass growth of the fungus for the isolation and identification of its metabolites was carried out in 1 L Erlenmeyer flasks (each flask containing 100 mL of distilled water were added to 100 g commercially available rice and kept overnight prior to autoclaving). The fungus was grown on rice solid medium (8 flasks) at room temperature under septic conditions for 4 weeks.

The seeds and roots of five adult healthy plants of P. cupana from each clonal cultivars CMU 871 (tolerant to anthracnose and oversprouting) and CMU 300 (tolerant to anthracnose and susceptible to oversprouting) were collected in November 2014 in the Embrapa (Brazilian Agricultural Research Company) experimental farms located in the cities of Manaus (2° 53′ 29.14″ S and 59° 58′ 39.90″ W, 99 m high) and Maues (3° 22′ 54″ S and 57° 42′ 55″ W, 18 m high), State of Amazonas, Brazil. The plant material was collected at the end of the dry season, when the accumulated rainfall and average temperature were respectively 196.0 mm and 28.46 ℃ in Manaus, and 272.3 mm and 28.17 ℃ in Maues. Five millimeter diameter plugs of each endophytic fungus were place at the center of Petri dishes (90 mm diameter), each containing 20 mL of PDA, and cultured for 13 days at 25 ℃.

The fungal strain Trichoderma sp. Xy24 was isolated from the leaves, stems and peels of mangrove plant X. granatum collected in Sanya district, Hainan province of China. The fungal strain was maintained on slants of modified potato dextrose agar (PDA) medium (potato 200 g, glucose 20 g, distilled water 1 L, KH₂PO₄ 3 g, MgSO₄ 0.75 g, vitamin B1 10 mg, agar 8.0 g, pH 6.0; the media were autoclaved at 115 ℃ for 30 min) at 4 ℃. Seed cultures were performed in Erlenmeyer flasks (250 mL) containing 100 mL of PDA liquid medium on a shaker at 150 rpm at 25 ℃ for 2 days, after that 5 mL seed cultures were inoculated into each 1000 mL flask with 300 mL medium and cultivated for 14 days (150 rpm, 25 ℃).

Isolation of endophytes: The leaves of 24 plant species were collected in the Amazon Rainforest biome, in Cayenne and Roura, French Guiana. After collection, the plant material was washed with sterile water and surface sterilised by sequential immersion in 70% aqueous ethanol (3 min), followed by 5% aqueous sodium hypochlorite (5 min) and finally by 70% aqueous ethanol (1 min). After these procedures, the leaves were rinsed with sterilised water. The residual sterilised water was incubated in Petri dishes to ensure that all the epiphytic microorganisms were eliminated. The surface-sterilised leaves were cut into 64 small pieces which were placed in 2 ml Eppendorf tubes containing 1 ml of potato dextrose agar medium (PDA, Fluka Analytical, Germany) at 26 ℃. Each individual hyphal tip of emerging fungi was removed and placed on a sterile PDA culture medium in 10 cm Petri dishes. The leaf fragments were cultured for a maximum of 5 months. All strains, including bacteria, were cultivated on solid PDA medium at 26 ℃ for 15 days, on 3 Petri dishes of 14 cm diameter (150 cm²). On the large scale, the microorganisms were cultivated under the identical conditions with 130 Petri dishes of 14 cm diameter (2 m²).