Mature fruits of a healthy and symptomless one year old plant of C. annum (Family: Solanaceae) were collected from Jammu (32° 43′ 58″ N, 74° 48′ 32″ E), India. An inoculum of A. alternata was prepared in Erlenmeyer flasks (500 ml) with potato dextrose broth (PDB) media (150 ml) incubated for 5 days in shaking (150 rpm) at 28 ℃ . Batch fermentation was performed in Erlenmeyer flasks (1L) on PDB, pH 5.5. Each flask containing 300 ml broth was inoculated with 10 percent of inoculum prepared and incubated at 28 ℃ in dark with shaking at 200 rpm for 7 days.

Endophytic fungi were isolated from fruits of M. dentata (Icacinaceae; sub-order Asterid). The species is a slow growing climber and is sparsely distributed in the Western Ghats, India. For each pure isolate, single hyphal tips were incubated in 250 ml conical flasks containing 50 ml of pre-sterilized PDB. Flasks were agitated at 200 rpm on a rotary shaker at 28 ℃ for 4 days.

Isolation of endophytes from Hypericum perforatum: Plant specimens of Hypericum perforatum were sampled at the Botanical garden of University of Szeged (N46.235, E20.159) in autumn. The leaf, stem, root and flower parts were separated, and these parts were examined for their fungal endophyte content. The plant materials were rinsed in running tap water to remove dust and debris and the specimens were cut into small segments of about 0.5 to 1 cm in length using a sterile blade. The plant segments were surface sterilized to kill the epiphytic microorganisms by sequentially immersing the plant material in 70% ethanol for 60 s, washing with sterile distilled water and then, steeping in 0.01% mercuric chloride (VWR) for 30 sec. Finally, the specimens were washed again with sterile distilled water 2-3 times and then allowed to dry on a sterile blotting paper. Each segment was placed onto the surface of potato dextrose agar (PDA; VWR) medium supplemented with ampicillin (50 mcg/mL) in a Petri dish. All plates were incubated at 25 ℃ for 5-10 days and were checked daily for the growth of fungal colonies. Pure isolates were obtained by picking up individual colonies from the plates and transferring them onto a fresh PDA medium where they were incubated at 25 ℃ for 10 days. Isolated endophytic fungi were cultured for 7 days at 25 ℃ in 50 ml PDB medium.

Isolation of endophytes from Hypericum perforatum: Plant specimens of Hypericum perforatum were sampled at the Botanical garden of University of Szeged (N46.235, E20.159) in autumn. The leaf, stem, root and flower parts were separated, and these parts were examined for their fungal endophyte content. The plant materials were rinsed in running tap water to remove dust and debris and the specimens were cut into small segments of about 0.5 to 1 cm in length using a sterile blade. The plant segments were surface sterilized to kill the epiphytic microorganisms by sequentially immersing the plant material in 70% ethanol for 60 s, washing with sterile distilled water and then, steeping in 0.01% mercuric chloride (VWR) for 30 sec. Finally, the specimens were washed again with sterile distilled water 2-3 times and then allowed to dry on a sterile blotting paper. Each segment was placed onto the surface of potato dextrose agar (PDA; VWR) medium supplemented with ampicillin (50 mcg/mL) in a Petri dish. All plates were incubated at 25 ℃ for 5-10 days and were checked daily for the growth of fungal colonies. Pure isolates were obtained by picking up individual colonies from the plates and transferring them onto a fresh PDA medium where they were incubated at 25 ℃ for 10 days. Isolated endophytic fungi were cultured for 7 days at 25 ℃ in 50 ml PDB medium.

The live yew branches were collected from branches of T wallichiana var. mairei (Lemee et H. Lev.) L. K. Fu et Nan Li growing in the Taihang Mountain, Henan Province, China in March 2014. The hypha with PDA pieces (approximately 4 mm × 4 mm) were inoculated into 60 mL PDB culture medium in culture flask for incubation at 120 rpm and 20 to 25 ℃ for 4 d. Five milliliters of liquid culture medium containing hyphae fung was inoculated into 60 mL PDB culture medium in culture flask and cultured at 120 rpm and 20 to 25 ℃ for 8 days. The uninoculated PDB culture medium in culture flask was taken as control.

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.

Healthy plant (Mirabilis jalapa L.) was collected from Dampa Tiger Reserve Forest [DTRF] (23° 44′ N 92° 39′ E), Mizoram, Northeast India during February, 2014. The cut ends were sealed with wax and were brought to the laboratory. Five mycelial agar plug of grown strain was inoculated in 2 L Erlenmeyer flasks containing 700 ml potato dextrose broth (PDB) media and incubated at 26 ℃ for three weeks.

Ketosynthase (KS) domain of polyketide synthases (PKS) type I and adenylation (A) domain of non-ribosomal peptide synthetases (NRPS) were detected in strain MJ31 which might play a role in antimicrobial activity. An expected 700 bp band of KS domain was detected by LC3 and LC5C primers which were responsible for the synthesis of partially reducing (PR) type PKSs. NRPS gene was also detected with the amplified product size of 300 bp.

The fungus P. variotii EN-291 was isolated from Grateloupia turuturu, a marine red alga collected from the coast of Qingdao, China, in May 2013. The fungal strain was statically fermented in a 1000-mL Erlenmeyer flask containing 300 mL of the PDB medium (potato dextrose broth: 2% mannitol, 1% glucose, 0.3% peptone, 0.5% yeast extract, and seawater added up to 300 mL, pH 6.5-7.0, adjusted with 10% NaOH/flask, 60 flasks) at room temperature for 30 days.

Samples of T. media were obtained from the campus of Huazhong University of Science and Technology (114° 31′ E, 30° 36′ N) in Hubei province in central China. Seven bark samples (0.5 × 3 cm) from yew trees were harvested and immediately transported to our laboratory and stored at 4 ℃. These endophytic fungi were cultured in 1-l Erlenmeyer flasks containing 300 ml potato dextrose liquid medium at 25 ℃ with 180 recycles/minute for 10 days.

Fresh young stems of A. flava were collected from Bogor Botanical Garden in 2008. Fresh AFKR-18 fungus grown on PDA medium was inoculated into 500 mL Erlenmeyer flasks containing 200 mL of PDB medium. After still incubation at room temperature for 3 weeks, the medium and biomass were homogenized and extracted three times with ethyl acetate-methanol (4:1) in equal volume.

Authenticated M. champaca was collected in the Chemistry Institute, Sao Paulo State University (UNESP), Araraquara, Sao Paulo, Brazil, in April of 2004. Endophytic fungi were isolated from the leaves, stems and roots of healthy, adult M. champaca, which was subjected to surface sterilization. C. gloeosporioides was cultured on a larger scale and inoculated into six Erlenmeyer flasks (4 L), each containing PDB medium (3.0 L), and incubated at 25 °C on rotary shakers at 150 rpm for 28 days.

The fungal strain Colletotrichum salsolae SCSIO 41021 (Glomerellaceae) was obtained from the inside of the stem of the mangrove plant Kandelia candel (L.) Druce (Rhizophoraceae) collected in Daya Bay, China (N22° 44′ 19.82″, E114° 31′ 58.18″) in March 2012. Strain stored on PDA slants at 4 ℃ was cultured on PDA agar plates and incubated for 7 days. Seed medium (potato 200 g, dextrose 20 g, NaCl 2.5 g, distilled water 1000 mL) in 50-mL Erlenmeyer flasks was inoculated with strain and incubated at 25 ℃ for 48 h on a rotating shaker (180 rpm). Production medium of 200-mL PDB in 1000 mL flasks was inoculated with 10 mL seed solution. Flasks were incubated at 25 ℃ under static stations and daylight.

The fungus was isolated as an endophyte from C. hirta obtained from Hawaii, USA. The culture was grown in PD broth (HiMedia, India) in an incubator shaker (New Brunswick) at 25 ℃ and 200 rpm for 15 days.

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.

Isolation of endophytes from Hypericum perforatum: Plant specimens of Hypericum perforatum were sampled at the Botanical garden of University of Szeged (N46.235, E20.159) in autumn. The leaf, stem, root and flower parts were separated, and these parts were examined for their fungal endophyte content. The plant materials were rinsed in running tap water to remove dust and debris and the specimens were cut into small segments of about 0.5 to 1 cm in length using a sterile blade. The plant segments were surface sterilized to kill the epiphytic microorganisms by sequentially immersing the plant material in 70% ethanol for 60 s, washing with sterile distilled water and then, steeping in 0.01% mercuric chloride (VWR) for 30 sec. Finally, the specimens were washed again with sterile distilled water 2-3 times and then allowed to dry on a sterile blotting paper. Each segment was placed onto the surface of potato dextrose agar (PDA; VWR) medium supplemented with ampicillin (50 mcg/mL) in a Petri dish. All plates were incubated at 25 ℃ for 5-10 days and were checked daily for the growth of fungal colonies. Pure isolates were obtained by picking up individual colonies from the plates and transferring them onto a fresh PDA medium where they were incubated at 25 ℃ for 10 days. Isolated endophytic fungi were cultured for 7 days at 25 ℃ in 50 ml PDB medium.

The endophytic fungus Eupenicillium sp. LG41 was isolated from the roots of Xanthium sibiricum (Asteraceae) collected from Taian, Shandong Province, People's Republic of China. This fungal strain was cultured on PDA medium at 28 ℃ for 1 week. Agar plugs were cut into small pieces, and 40 pieces were selected to inoculate 40 Erlenmeyer flasks (500 mL) each containing 250 mL of PDB. The cultures were incubated at 28 ℃ on a rotary shaker (150 rpm) for 28 days. The endophytic fungus was further fermented in 35 Erlenmeyer flasks (500 mL) each containing 250 mL of PDB supplemented with 15 mg/100 mL of nicotinamide. The cultures were incubated at 28 ℃ on a rotary shaker (150 rpm) for 28 days.

The fungus used in this study was isolated from stem cuttings of C. roseus plant collected from the nursery of the Indian Institute of Science, Bangalore, India. The endophytic fungus was grown on PDA plates for about 10 days. Three 5 mm agar plugs containing mycelium of Eutypella spp were transferred to 400 ml of potato dextrose broth, in a 1 L conical flask and incubated at 25 ℃ under stationary condition for 21 days.

Endophytic fungi were isolated from fruits of M. dentata (Icacinaceae; sub-order Asterid). The species is a slow growing climber and is sparsely distributed in the Western Ghats, India. For each pure isolate, single hyphal tips were incubated in 250 ml conical flasks containing 50 ml of pre-sterilized PDB. Flasks were agitated at 200 rpm on a rotary shaker at 28 ℃ for 4 days.

Strain BLH51 used in this study was 1 of 55 endophytic fungi isolated from the leaves of M. cordata from Dabie Mountain, China. The endophytic fungi isolates were inoculated, respectively, into 500 mL Erlenmeyer flasks containing 100 mL of potato dextrose liquid medium and cultured at 28 °C with 200 recycles/minute for 10 days in a rotary shaker.

The fungus was obtained from the fresh leaves of Euphorbia sieboldiana, which were collected from the campus of China Pharmaceutical University, Nanjing, Jiangsu, People's Republic of China, in April 2013. After cultivation on potato dextrose agar (PDA) at 28 °C for 7 days, two pieces of the agar (about 1.0 cm³) were added to an Erlenmeyer flask (500 mL) with 150 mL of potato dextrose liquid medium, and the flask was incubated on a rotary shaker at 28 °C and 150 rpm for 5 days to prepare seed culture. Solid fermentation was carried out in 12 Erlenmeyer flasks (500 mL) each containing 120 g of sterilized rice and 10 mL of seed culture, and the flaks were cultivated at 28 °C for 30 days.

The intact plant was collected from an altitude of 2600 m in the Taibai Mountains of Shaanxi, China in August 2009. The plant was identified morphologically and phylogenetically to be S. hexandrum. Strain TW5 was inoculated into 300 flasks (500 mL) containing 200 mL of fermentation medium and incubated at 26 ℃ on a reciprocal shaker at 180 rpm. After 7-d incubation, 60 L of culture liquid containing 4250 g of mycelia (wet weight) was obtained.

Healthy leaves of the medicinal plant S. china were collected from LongQuan County of Zhejiang Province, China. Strain ZJLQ129 was grown in 1 L Erlenmeyer flasks containing 500 mL potato dextrose broth (PDB, 40 L in total) medium. The flasks were cultured without shaking for 14 days at room temperature.

Fungal cultures were isolated from freshly harvested Corylus avellana "Barcelona" nuts from Aurora, Oregon, USA. A 1 week-old sporulating culture on PDA was rinsed with 20 mL of sterile water containing 1 drop of Tween-20. Two mL of the spore solution with an absorbance of about 0.8 at 600 nm was added to each of 6 liters of potato dextrose broth (PDB, DIFCO). Broth cultures were shaken at 20 &#8451 and 100 rpm for 2 weeks. On Day 14, when the amount of reducing sugars in the cultures was no longer detectable using glucose test strips, 100 µL of methyl jasmonate and 0.172 g/L filter-sterilized phenylalanine were added to each flask, and shaking was resumed. The cultures were harvested on day 24.

The healthy leaves of Tapiscia sinensis Oliv. were collected in the Shennongjia National Forest Park. The fungus (NO. 24) was cultured in 50 L PDB liquid medium with 250,500 mL-Erlenmeyer flasks on the electronic oscillator under 28 ℃ with the speed at 120 r/min for 20 days.

Pestalotiopsis foedan was isolated from the branches of Bruguiera sexangula in Hainan, P. R. China, in April 2008, identified by Prof. Jing-Ze Zhang, and assigned the accession number L444. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 30 °C for 5 d, and then inoculated into 500 ml Erlenmeyer flasks containing 100 ml of PDB. After 7 d of incubation at 30 °C on rotary shakers at 160 rpm, 20 ml of culture liquid was transferred as inoculum into 1000 ml Erlenmeyer flasks containing 200 ml of PDB. Fermentation was carried out at 30 °C on rotary shakers at 150 rpm for 22 d.

Healthy shoots of D. officinale plants were collected in Yandang Mountain, Zhejiang Province, PR China. DO14 was inoculated in Erlenmeyer flasks with potato dextrose broth (PDB) and incubated on a rotary shaker (180 rpm) for 7 days at 28 °C.

Fungal strain G536 was isolated from surface sterilized twigs of paw paw (A. triloba (L.) Dunal, Annonaceae) collected from Pfafftown, NC, USA (36°09′ 58.8″ N 80°24′18.6″W). In preparation for chemical extraction, G536 was grown on rice, as this has been shown to be an efficient medium for the production of secondary metabolites in culture. To make seed cultures for inoculating rice, a piece of fresh culture grown in potato dextrose (PD) (Difco) or 2% malt extract (ME) (Difco) media was excised from the leading edge of the colony and transferred to a liquid seed medium containing 2% soy peptone, 2% dextrose, and 1% yeast extract (YESD; 5 g of yeast extract, 10 g of soy peptone, and 10 g of d-glucose in 500 ml distilled water). Following incubation for 7 days at 22°C with agitation, the culture was used to inoculate 10 g of rice media prepared using 30 ml of distilled H₂O and autoclaved in a 250 ml Erlenmeyer flask. This screener scale fermentation was incubated at 22°C for 14-21 days prior to chemical extraction. For large-scale production of fungal cultures, four 250 ml Erlenmeyer flasks containing 10 g of rice were inoculated using one seed culture for each flask.

Endophytic fungi were isolated from fruits of M. dentata (Icacinaceae; sub-order Asterid). The species is a slow growing climber and is sparsely distributed in the Western Ghats, India. For each pure isolate, single hyphal tips were incubated in 250 ml conical flasks containing 50 ml of pre-sterilized PDB. Flasks were agitated at 200 rpm on a rotary shaker at 28 ℃ for 4 days.

Fungal Material. The fungus was isolated from surface-sterilized fresh stems of an apparently healthy Aconitum carmichaeli collected in August 2008 in Huize County, Yunnan Province, P. R. China. The fungal strain was cultured on slants of PDA at 28 ℃ for 7 d. Then, the fresh mycelium was inoculated into 20x500-ml Erlenmeyer flasks each containing 120 ml of PDB medium (200 g potato and 20 g glucose in 1 l of H₂O). After 4 d of the incubation at 28 ℃ on a rotary shaker at 200 r.p.m., a 20-ml culture liquid was transferred as seed into each of a total of 100 1-l Erlenmeyer flasks containing 250 ml of PDB medium. The following cultivation was kept for 7 d at 28 ℃ at 200 r.p.m. on a rotary shaker.

Healthy, asymptomatic foliage of one individual of Platycladus orientalis was harvested at the Campus Arborteum at the University of Arizona (32.231° N, 110.952° W, elevation 787 m; mean annual temperature = 20.2 &#8451) in April 2005. This site, which is located within the Sonoran Desert bioregion, is relatively xeric, receiving an average of 30.5 cm of precipitation annually. A culture of P. spinarum (BA 9149b-4) grown in PDB (2 L) was filtered, and the filtrate was extracted with EtOAc (5 × 500 mL).

The fungus P. acaciicola was isolated from the healthy roots of a mangrove tree, B. gymnorrhiza, collected in November 2012 at The Mangrove Forest Learning and Development Center 2, Samut Sakhon province, Thailand. The fungus P. acaciicola was cultured on Potato Dextrose Agar (PDA) (HiMedia Laboratories, Mumbai, India) containing 10% of seawater (collected from Bang Saen Beach, Chonburi Province) for 1 week. The agar was then inoculated into 500 mL of Potato Dextrose Broth (PDB) (HiMedia Laboratories, Mumbai, India) containing 10% of seawater. After incubation for 21 days, broth (total volume of 500 mL) and fungal mycelia were separated by filtration using filter paper (Whatman, Grade 1). The second batch of fungal culture was a scaled-up cultivation of the fungus P. acaciicola. The fungus was cultured on PDA prepared in a mixture of seawater and distilled H₂O (10% seawater). After incubation for 1 week at 30 &#8451, the agar was then inoculated into 1000 mL Erlenmeyer flasks (forty flasks), each containing 250 mL of PDB prepared in 10% seawater. After incubation for 21 days, fungal culture was filtered to separate mycelia and broth (total volume of 10 L).

Stems (0.5-1.0 cm in diameter) of Kennedia nigriscans ) were obtained near the Aboriginal community of Manyallaluk, SE of Katherine, Northern Territory, Australia, at 14° 16′ 352″ S and 132° 49′ 750″ E. Several small blocks of PDA containing the streptomycete were inoculated into 500 ml PD broth in a 2 l Erlenmeyer flask and incubated for 3 weeks at 23 ℃ without shaking.

The fungus used in this study was one of 45 endophytic fungi isolated from the inner bark of T. mairei obtained in Fujian province, southeast China.The endophytic fungus strain TF5 was grown in 2-lErlenmeyer asks containing 500 ml potato dextrose liquidmedium. The fungus was incubated at 25 &#8451 without agitation for 21 days.

Healthy, symptomless leaves of Aegle marmelos were collected from Yamuna Nagar district of Haryana, India. To prepare the inoculum of the fungal culture, Erlenmeyer conical flasks of capacity 500 ml containing potato dextrose broth (PDB, 200 ml) were used. The flasks were inoculated with fungal agar plugs, kept in shaking incubator at 28 ℃ with 150 rpm for 3 days. The prepared inoculums (10%) was used to obtain large amount of fermented culture broths in Erlenmeyer flasks (1 l) containing PDB (350 ml). The flasks were kept in shaking incubator at 28 ℃ with 150 rpm and dark for 10 days.

The endophytic fungus Coniothyrium sp., internal strain No. zw86, was isolated from the plant Salsola oppostifolia, growing on Gomera, in the Canary Islands. It was cultivated on 12 l of 5% w/v biomalt solid agar medium at room temperature for 28 days.