Molecular phylogeny of orchids mycorrhiza isolated from native tropical orchids in Indonesia

@#Aims: Mycorrhiza has an important role as a biocontrol agent. Its association with Phalaenopsis amabilis was molecularly identified through rDNA-ITS sequence analysis. The aims of the study were to identify molecular of orchids mycorrhiza isolate from native tropical orchids in Indonesia, conducted as one of native orchid conservation efforts in Indonesia. Methodology and results: One group of Ceratobasidium were isolated from the root of orchid plant in Yogyakarta based on morphological and microscopical analysis. The results of molecular analysis showed 600-750 bp of DNA products located on the ITS1-5.8S-ITS4 region. The sequenced products showed insertion and substitution occurances, which may result in strain diversity and possible variation. Reconstruction of phylogenetic trees using Maximum Parsimony and Bootstrap-1000 approach showed showed the Indonesian isolate is at the basal clade and already far apart from the other isolates. Conclusion, significance and impact of study: Isolate Ceratobasidium from Yogyakarta, Indonesia successfully isolated based on identification of rDNA-ITS sequences. Results of this study were expected to become the basic information in an effort of native orchid cultivation and protection against infectious diseases in Indonesia. The study was the first to report regarding Ceratobasidium isolated from native tropical orchids in Indonesia.

Mycorrhizal Diversity Associated to Liparis japonica (Miq.) Maxim. in China

Orchidaceae (Orchidaceae) is the second largest family of angiosperms. It’s the "flagship" group in plant protection. The existence of orchid plant is closely related to mycorrhizal fungi. The relationship between orchids and their symbiotic mycorrhizal fungi is a benefit to the protection and population restoration of orchids. Aims: The research was aimed to study molecular identification about 15 strains of mycorrhizal fungi from 6 plots by rDNA ITS technology in order to understand and utilise the mycorrhizal fungi of Liparis japonica (Miq.) Maxim. Study Design: The mycorrhizal fungi collected from different geographical locations were isolated and purified from the mycorrhizal fungi symbiotic with Liparis japonica in Northeast China, which were identified by rDNA ITS, meanwhile computed evolutionary distance and constructed the phylogenetic tree. Place and Duration of Study: In 2017, the root segments of Liparis japonica were collected separately from Qianshan, Changbaishan, Gaoguan, Guanmenshan, Dongling, Daqinggou. Methodology: Fifteen strains of mycorrhizal fungi collected from six plots were identified by rDNA ITS. Using DNAMAN software to analyse, the pairwise homology was compared by using the optimal global sequencing option. The evolutionary distances of fifteen strains were calculated by MEGA (Molecular Evolutionary Genetics Analysis) software package and their phylogenetic trees were constructed by neighbour-joining method. Results: With primers ITS1 and ITS4, the 15 mycorrhizal fungi strains of rDNA ITS got about 600 bp length. ITS length was about 582-613 bp, in which ITS1 length was about 177-190 bp, and ITS2 length was 246-273 bp. The mycorrhizal fungi strains were highly homology separated from one plot, mostly above 90%. The plots from the south to the north were as follows: Qianshan, Guanmenshan, Gaoguan, Dongling, Changbaishan, Daqinggou in China. Fifteen strains after separated and purified were identified to be the Epulorhiza of Orchid Rhizoctonia blasted with Genbank. The homology of the strains gradually decreased affected by the difference of the north and the south, namely there was an increasing trend of diversity from south to north. Conclusion: The homology of mycorrhizal fungi from one plot was higher because of the same soil environment and climate environment and so on, and strain type was single. Under the influence of microclimate in Northeast China, the homology of strains decreased gradually in the sample area, that is, the diversity gradually increased from the south to the north.

Taxonomy of the Golovinomyces cynoglossi Complex (Erysiphales, Ascomycota) Disentangled by Phylogenetic Analyses and Reassessments of Morphological Traits

The name Golovinomyces cynoglossi s. lat. is traditionally applied to a complex of morphologically similar powdery mildews on hosts of the plant family Boraginaceae. The current species-level taxonomy within this complex is ambiguous due to the lack of phylogenetic examinations. The present study applied phylogenetic methods to clarify the taxonomy of G. cynoglossi s. lat. Phylogenetic analysis of rDNA ITS sequences retrieved from Asian, European and North American specimens revealed that G. cynoglossi s. lat. collections from different hosts involved several species in five clearly separated lineages. Clade I consists primarily of Golovinomyces cynoglossi s. str. on Cynoglossum. Clade III consists of Golovinomyces sequences retrieved from the host genera Symphytum and Pulmonaria. The taxa within clade III are now assigned to G. asperifoliorum comb. nov. Clade V encompasses G. cynoglossi s. lat. on the host genera Bothriospermum, Buglossoides, Echium, Myosotis, and Trigonotis. The taxa within clade V are now assigned to G. asperifolii comb. nov. The species concerned in this study were lecto- and epitypified to stabilize their nomenclature.

Three New Recorded Species of the Physalacriaceae on Ulleung Island, Korea

Most known species in the Physalacriaceae are saprotrophs that grow on decaying leaves and wood, and approximately 21 genera in the Physalacriaceae have been reported worldwide. During an ongoing survey of indigenous fungi in Korea, four specimens belonging to the Physalacriaceae were collected on Ulleung Island. These specimens were identified as three species based on morphological characteristics and molecular analysis of rDNA-internal transcribed spacer sequences. Three species in three genera were shown to be new records in Korea: Hymenopellis orientalis, Paraxerula hongoi, and Ponticulomyces orientalis. The latter two are the first records of these genera in Korea. In this study, we provide detailed morphological descriptions of these species and describe their phylogenetic position within the Physalacriaceae.

rDNA-ITS and 28 S rDNA D1/D2 sequence analysis of downy mildew pathogen from Aconitum carmichaeli / 中草药

Objective: To define sequences about rDNA-ITS and 28 S rDNA D1/D2 of Peronospora aconiti separated from cultivated Aconitum carmichaeli in Jiangyou area of Sichuan province and provide a theoretical basis for the diagnosis and prevention of downy mildew disease. Methods: Spores and hyphae of P. aconiti from diseased plants were collected and total genomic DNA of pathogen were extracted and then rDNA-ITS and 28 S rDNA D1/D2 fragment were amplified and sequenced. According to the above results, the abutment (Neighbor-joining, NJ) phylogenetic tree of pathogen was constructed and analyzed. Results: The rDNA-ITS and 28 S rDNA D1/D2 sequences of P. aconiti were sequenced and compared according to the database from NCBI. Compared that with P. pulveracea and P. aparines, the similarity of rDNA-ITS sequences of P. aconiti was 94%. The similarity of 28 S rDNA D1/D2 sequences of P. aconiti was 97% compared that with P. pulveracea, P. ficariae and P. bulbocapni. Conclusion: The results of morphological identification of downy mildew pathogen separated from A. carmichaeli are consistent with those from molecular identification (rDNA-ITS and 28 S rDNA D1/D2 sequences) and the pathogen of Aconitum downy mildew should be P. aconiti. Therefore, rDNA-ITS and 28 S rDNA D1/D2 sequences constructed in this paper can be used to identify downy mildew pathogen from Aconitum carmichaeli Debx.

Distribution and phylogenetic analysis of endophytic fungi in stems and leaves of Desmos chinensis based on histochemistry and rDNA ITS sequence / 中国药学杂志

OBJECTIVE: To study the distribution and phylogenesis of endophytic fungi in the stems and leaves of Desmos chinensis. METHODS: Permanent paraffin-cut section, optical microscope photography, and histochemistry were used to observe the distribution of endophytic fungi in the stems and leaves of Desmos chinensis. Plate isolation method was used to separate and cultivate the endophytic fungi. The genomic DNA of endophytic fungi were extracted, then the rDNA ITS region was amplified using polymerase chain reaction (PCR). The PCR products were digested by Hae III and Hha I restriction enzymes to determine the species and genotypes, and sequenced according to the digestion genotyping results. Nucleotide sequences of the rDNA ITS of endophytic fungi were used for the identification and phylogenetic analysis. RESULTS: Endophytic fungi mainly existed in the parenchyma cells of the phloem in the stems, and in the parenchyma cells, palisade tissue or sponge tissue of the leaves. Fourteen strains of endophytic fungi were isolated from the stems and leaves. Four different genotypes and their sequences were obtained from RFLP analysis and sequencing. These strains were identified as Guignardia mangiferae, Colletotrichum gloeosporioides, and Phomopsis spp. The dominant group of those endophytic strains was Phomopsis, represented by 8 strains and accounting for 57.1% of all. CONCLUSION: The endophytic fungi distribute in both stems and leaves of Desmos chinensis without obvious tissue specificity. The result of the phylogenetic analyses with rDNA ITS sequences reveals the phylogenetic relationships of endophytic fungi in Desmos chinensis.

Sequence analysis of ribosomal DNA internal transcribed spacer 2 of sym-patric populations of Anopheles sinensis of different feeding preferences / 中国血吸虫病防治杂志

Objective To investigate the existence of genetic divergence of sympatric populations of Anopheles sinensis of different feeding preferences based on the rDNA-ITS2 sequence differences. Methods A large number of wild anopheles popu-lations were trapped all night by man-baited net and calf-baited net that had been set up between high-density natural villages of An. sinensis populations and vector-breeding sites,from which two groups of An. sinensis were separated by morphological iden-tification and brought back to the lab for conventional breeding. A large closed greenhouse which temperature and humidity was appropriate was selected as research settings of mark-release-recapture methods by female mosquitoes ,in the center of which above An. sinensis populations baited by man and calf and respectively correspondingly marked by red and yellow phosphors were released in together,in each side of which An. sinensis were recaptured simultaneously by man-baited net and calf-baited net. An. sinensis populations trapped by man twice were brought back to the lab and bred with man-blood,correspondingly ones trapped by calf with calf-blood. Man-preferring and calf-preferring strains were screened respectively from An. sinensis which had been baited by man and calf by the mark-release-recapture methods after parent and F1 mosquitoes,and sequencing and aligning of both rDNA-ITS2 were conducted via PCR amplification. Results The recapture ratios of wild parental mosquitoes An. sinensis of man-preferring group by man-baited net and calf-baited net were 54.07%(339/627)and 45.93%(288/627)re-spectively,and ones of calf-preferring group by man-baited net and calf-baited net were 58.01%(409/705)and 41.99%(296/705)respectively. Two groups of parental mosquitoes trended towards selecting the original blood hosts in host-seeking prefer-ence(χ2=19.42,P<0.01). The recapture ratios of F1 mosquitoes An. sinensis of man-preferring group by man-baited net and calf-baited net were 63.43%(765/1 206)and 36.57%(441/1 206),and ones of calf-preferring group by man-baited net and calf-baited net were 68.22%(1 039/1 523)and 31.78%(484/1 523). Two groups of F1 mosquitoes had more significant characteris-tics of selecting the original blood hosts in host-seeking preference(χ2=271.69,P<0.01)and showed the genetic differentia-tion phenomenon,but the results of sequencing and aligning of the rDNA-ITS2 via PCR amplification showed no difference in base sequence between the two strains and both were 469 bp. Conclusions The genetic divergence based on the rDNA-ITS2 se-quence does not happen in An. sinensis sympatric populations of different feeding preferences.

Isolation and identification for strains from wild Phellinus and analysis on its secondary metabolites / 中草药

Objective: To isolate and identify the strains from wild Phellinus linteus and analyze its secondary metabolites. Methods: Strains isolated and purified were investigated by optical microscope to observe the characteristics of mycelia, ITS sequence was used for molecular identification and its secondary metabolite content was analyzed by chemical colorimetry. Results: The morphological characteristics of mycelia from pinus, Morus alba, Syringa reticulata, and populus were the same. The growth rate of four flamentous colonies and their colors were different, wherein the growth rate of the colony from M. alba was the fastest, up to 0.47 cm/d. Four strains all belonged to genus Phellinus sp, where strain from pinus was identified as P. pini, M. alba was P. linteus, S. reticulata and populus were P. baumii. The species of the secondary metabolites in the sporocarp and mycelia of Phellinus were the same, but the content was different. For the sporocarp, the highest polysaccharide content was in the sporocarp from S. reticulata with the content of 98.20 mg/g, the highest flavonoids, terpenoids, and polyphenols contents were all in the sporocarp from poplar with the contents of 548.49, 1.48, and 33.70 mg/g, respectively. For mycelia, the highest polysaccharide, flavonoids, terpenoids, and polyphenols contents were all in the mycelia of M. alba, the contents were 259.64, 223.11, 43.78, and 24.80 mg/L, respectively. Additionally, the content of terpenoids in the mycelia was higher than that of the sporocarp. Among them, the content of terpenoids in the mycelia from M. alba was about 7 times as that in fruit body. Conclusion: There are no differerences amony form of mycelia from four strains of P. linteus. rDNA ITS sequence analysis could be used for the identification of strains. The species four Phellinus strains and its secondary metabolite content are clarified.

Pythium insidiosum Thai isolates: molecular phylogenetic analysis

Background: Pythium insidiosum is an oomycete that infects both humans and animals, leading to a life-threatening infectious disease called “pythiosis”. Animal pythiosis presents with lesions of the skin, gastrointestinal tract, lung and bone, whereas human pythiosis presents with two common clinical forms, vascular pythiosis involving arteries, and ocular pythiosis involving the eye. Pythiosis in humans has been reported exclusively from Thailand. The disease in animals has been found around the world, but its occurrence has never been reported from Thailand.Objective: To group P. insidiosum based on molecular phylogenetic analysis, investigating correlation between phylogenetic group, geographic distribution, and host specificity of this pathogen.Methods: 113 rDNA internal transcribed spacer sequences of P. insidiosum were also obtained for phylogenetic analyses. These included 32 human isolates and 59 environmental isolates from Thailand, and four additional human isolates and 18 animal isolates from around the world.Results: P. insidiosum existed in three distinct clades in accordance with geographic distribution; clade-I contained American isolates, clade-II contained Asian and Australian isolates, and clade-III contained mainly Thai isolates. The Thai isolates existed only in clade-II and clade-III.Conclusion: There were two major subpopulations of P. insidiosum in Thailand. There were no correlation between the two Thai subpopulations of P. insidiosum and geographic regions or host specificity.

Identification and Characterization of the Causal Organism of Gummy Stem Blight in the Muskmelon (Cucumis melo L.)

Gummy stem blight is a major foliar disease of muskmelon (Cucumis melo L.). In this study, morphological characteristics and rDNA internal transcribed spacer (ITS) sequences were analyzed to identify the causal organism of this disease. Morphological examination of the Jeonbuk isolate revealed that the percentage of monoseptal conidia ranged from 0% to 10%, and the average length x width of the conidia was 70 (+/- 0.96) x 32.0 (+/- 0.15) microm on potato dextrose agar. The BLAST analysis showed nucleotide gaps of 1/494, 2/492, and 1/478 with identities of 485/492 (98%), 492/494 (99%), 491/494 (99%), and 476/478 (99%). The similarity in sequence identity between the rDNA ITS region of the Jeonbuk isolate and other Didymella bryoniae from BLAST searches of GenBank was 100% and was 95.0% within the group. Nucleotide sequences of the rDNA ITS region from pure culture ranged from 98.2% to 99.8%. Phylogenetic analysis with related species of D. bryoniae revealed that D. bryoniae is a monophyletic group distinguishable from other Didymella spp., including Ascochyta pinodes, Mycosphaerella pinodes, M. zeae-maydis, D. pinodes, D. applanata, D. exigua, D. rabiei, D. lentis, D. fabae, and D. vitalbina. Phylogenetic analysis, based on rDNA ITS sequence, clearly distinguished D. bryoniae and Didymella spp. from the 10 other species studied. This study identified the Jeonbuk isolate to be D. bryoniae.

Utility of the rDNA-ITS2-PCR assay in detecting the life stages of species S of Anopheles fluviatilis complex.

Background & objectives: Anopheles fluviatilis, which ranks second among the major malarial vectors in India occurs as a complex of three morphologically identical species (species S, T and U) of which only species S is a vector. Hence, it becomes pertinent to have a method for the detection of this vector species under field conditions to map the distribution of this vector. An rDNA-ITS2-PCR assay has been developed earlier for species S of this complex using female adult specimens. In order to widen the range of samples on which this technique can be employed, the utility of this PCR assay in detecting different life stages/gender/parts of the vector species was studied. Also, its reliability in detecting a single species S in pools of species T was studied. Methods: Mosquitoes were collected from Malkangiri and Koraput districts of Orissa State where species S and T of this complex are reported. The wild caught fed females, after egg laying were subjected to PCR assay for species identification. The F1 progeny of a few PCR identified specimens was raised and samples at larval, pupal and adult stages were used for PCR assay. Single adult specimen of species S was added to pools containing different numbers of adults of species T and the pools were subjected to DNA extraction and PCR assay. Results: The PCR assay could detect species S from pure DNA extracts of the immature stages and crude DNA extracts of parts of adult/whole adult mosquito of either gender. Crude DNA extracts of pools of mosquitoes had to be diluted and used in order to obtain the species diagnostic fragment. Interpretation & conclusions: The rDNA-ITS2-PCR assay producing an amplicon of 350 bp. diagnostic for species S, could detect all stages/gender. Any part of the adult can be used for species identification. Further, a single adult of species S in pools of as many as 99 adults of species T could be detected. Application of this PCR assay will be useful in mapping the distribution of species S, an important malarial vector.

Molecular Identification of Anopheles maculatus Complex from China / 中国寄生虫学与寄生虫病杂志

Objective To establish the molecular identification of five members in Anopheles maculatus complex from China. Methods Different rDNA-ITS2 regions of An. maculatus complex were sequenced and analyzed. The species specific primers were designed, and PCR assay was used for the identification. Results The length and GC contents of ITS2 were 328 bp, 58.54% in An. pseudowillmori, 330 bp, 57.85% in An. maculatus, 337 bp, 59.05% in An. willmori, 334 bp, 58.68% in An. dravidicus, and 338 bp, 57.69% in An. sawadwongporni, respectively. The intra-species ITS2 sequences were conservative. The ranges of divergence level among five members were from 9.7% to 18.9% . Five distinct specific fragments were amplified by PCR assay using five species specific primers and 5. 8S primer. The length was 119, 186, 231, 327 and 406 bp respectively. Conclusion The diagnostic PCR assay based on ITS2 divergence to distinguish five members of An. maculatus complex was simple and reliable.

Ribosomal DNA-ITS sequence analysis and molecular identification of Morinda officinalis and its counterfeit species / 中草药

Objective To compare the rDNA-ITS differentiation and its regulation between Morinda (officinalis) and its counterfeit species, and provide DNA molecular markers for the fingerprint identification of them. Methods The rDNA-ITS regions of M. officinalis and its counterfeit species were amplified and sequenced, then analyzed by means of CLUSTRAL X and MEGA softwares. Results The internal transcribed spacers (ITS) including ITS1, 5.8S, ITS2, and partial 18S and 26S were determined. In DNA DIST analysis, the range of diversity among M. officinalis and M. shughuaeusis, M. umbellata was (2.9%-)5.8% and 2.9%-4.2% based on ITS1 and ITS2; the range of diversity between M. officinalis and Damnacanthus indicus was 21.2% and 18.9% based on ITS1 and ITS2. Phylogenetic tree based on ITS and 5.8S sequence data indicated the M. umbellata and M. shuanghuaensis were closely related then with M. officinalis, while D. indicus was monophyletic group. Conclusion The rDNA-ITS sequence is a better molecular marker for idertification of M. officinalis and its counterfeit species.

Identification of Trapa L. plants along middle-low reaches of Changjiang River by analyzing their DNA sequences / 中草药

Object To analyze the rDNA ITS sequences between wi ld plants and cultivars of Trapa L. and study the utility in p hylogenesis and identification of these two groups. Methods The ITS gene fragments were PCR amplified and sequenced. The rDNA ITS regions w ere analyzed by means of the software of Clustal and Mega 2.0. Result s The rDNA sequences of 234-236 bp ITS1, 220-221 bp ITS2 gene fragment , and 5.8 S rDNA for 164 bp evenly were obtained from ten populations of Trapa L. The intraspecific substitution varies from 0.22% to 2. 94%. The variable sites are 16 while informative sites are six. The phylogenet ic tree based on ITS data was set up by NJ method. Conclusion ITS sequence is a pretty good molecular marker which can identify wild plants of Trapa L. from their cultivars. Diversity of ITS in differen t populations is less at intraspecific level. It is infered that the plants of Trapa L. may be derived from the same population of one species .

Sequence analysis of rDNA ITS of de-virus seedlings of Pseudostellaria heterophylla / 中草药

Objective To study rDNA ITS sequence similarities and differences between the de-virus seedlings and outside seedlings of Pseudostellaria heterophylla. Methods The sequences of ITS region (including ITS1, ITS2, and 5.8S) from nine de-virus seedlings and four outside seedlings were studied by the method of DNA sequence analysis. Results There were the same sequences in 5.8S region between the de-virus seedlings and outside seedlings of P. heterophylla. Variable sites lay in the initial position of ITS1 and termination of ITS2 basically. The de-virus seedlings from Liyang (Jiangsu Province) and Xuancheng (Anhui Province) had more variable sites. Phylogenetic dendrogram based on ITS was constructed by using UPGMA methods, which showed the inherent relation at the level of molecular biology. Conclusion The analysis of rDNA ITS of P. heterophylla has further clarified the inherit stability of de-virus seedlings at the molecular level.