Insidensi dan Sebaran Tungau Hama pada Pepaya di Pulau Lombok (Incidence and Distribution of Mites on Papaya in Lombok Island)

<p>Tungau hama merupakan salah satu penyebab penurunan produksi pepaya di Pulau Lombok. Penelitian bertujuan menentukan insidensi, sebaran, dan identitas tungau hama pepaya di Pulau Lombok. Sebaran ditentukan berdasarkan insidensi tungau hama pada 50 lokasi pengambilan contoh dan tungau hama diidentifikasi secara morfologi dan molekuler berdasarkan runutan rDNA ITS2. Hasil penelitian menunjukkan terdapat 12 spesies tungau berdasarkan identifikasi morfologi, yaitu Aculops pelekassi, Calacarus carinatus, Brevipalpus californicus, B. obovatus, B. phoenicis, Tenuipalpus pasificus, Eutetranychus africanus, Panonychus citri, Tetranychus fijiensis, T. kanzawai, T. piercei, dan Tarsonemus bilobatus dengan insidensi berkisar antara 2–72%. Di antara spesies yang ditemukan, P. citri merupakan tungau hama dengan sebaran dan insidensi tertinggi (72%). Hasil analisis persebaran menunjukkan bahwa keanekaragaman spesies tungau hama pada tanaman pepaya di Pulau Lombok adalah tinggi dengan tingkat dominansi rendah dan tingkat kemerataan spesies yang tinggi. Uji PCR dan analisis runutan DNA berhasil mendeteksi dan mengidentifikasi enam spesies tungau hama, yaitu T. piercei, T. kanzawai, E. africanus, dan P. citri (Tetranychidae), pada 500–600 pb serta B. californicus dan B. phoenicis (Tenuipalpidae) pada 600–700 pb. Similaritas tertinggi ditemukan pada T. piercei dan T. kanzawai (100%). Ini merupakan laporan pertama keberadaan B. californicus sebagai hama pada tanaman pepaya di Pulau Lombok.</p><p><strong>Keywords</strong></p><p>Frekuensi kemunculan; PCR; Perunutan DNA; Sebaran</p><p><strong>Abstract</strong></p><p>Mites are one obstacle of papaya production in Lombok Island. Thus, the aim of research was to determine incidence, distribution and identity of mites on papaya plant in Lombok Island. Distribution is determined based on incidence of in 50 sampling area, while mites identified morphological and molecularly based on rDNA ITS2. This studies revealed that there were 12 species of mites based on morphological, namely Aculops pelekassi, Calacarus carinatus, Brevipalpus californicus, B. obovatus, B. phoenicis, Tenuipalpus pacificus Eutetranychus africanus, Panonychus citri, Tetranychus fijiensis, T. kanzawai, and T. pierce with an incidence ranging 2-72%. Among species found, P. citri has the highest distribution and incidence of 72%. The results of the distribution analysis showed that diversity of mite species was high, with low dominance and high evenness. PCR assay successfully amplified DNA of six species, namely T. piercei, T. kanzawai, E. africanus, P. citri with the DNA size of 500-600 bp and B. californicus, B. phoenicis with the DNA size of 600-700 bp. The highest similarity species was found on T .piercei and T. kanzawai (100%). This was the first report of B. californicus infestating on papaya in Lombok.</p>

Molecular phylogeny of the Anopheles hyrcanus group (Diptera: Culicidae) based on rDNA–ITS2 and mtDNA–COII

Background The Anopheles hyrcanus group, which includes 25 species, is widely distributed in the Oriental and Palaearctic regions. Given the difficulty in identifying cryptic or sibling species based on their morphological characteristics, molecular identification is regarded as an important complementary approach to traditional morphological taxonomy. The aim of this study was to reconstruct the phylogeny of the Hyrcanus group using DNA barcoding markers in order to determine the phylogenetic correlations of closely related taxa and to compare these markers in terms of identification efficiency and genetic divergence among species. Methods Based on data extracted from the GenBank database and data from the present study, we used 399 rDNA–ITS2 sequences of 19 species and 392 mtDNA–COII sequences of 14 species to reconstruct the molecular phylogeny of the Hyrcanus group across its worldwide range. We also compared the performance of rDNA–ITS2 against that of mtDNA–COII to assess the genetic divergence of closely related species within the Hyrcanus group. Results Average interspecific divergence for the rDNA–ITS2 sequence (0.376) was 125-fold higher than the average intraspecies divergence (0.003), and average interspecific divergence for the mtDNA–COII sequence (0.055) was eightfold higher than the average intraspecies divergence (0.007). The barcoding gap ranged from 0.015 to 0.073 for rDNA–ITS2, and from 0.017 to 0.025 for mtDNA–COII. Two sets of closely related species, namely, Anophels lesteri and An. paraliae, and An. sinensis, An. belenrae and An. kleini, were resolved by rDNA–ITS2. In contrast, the relationship of An. sinensis/An. belenrae/An. kleini was poorly defined in the COII tree. The neutrality test and mismatch distribution revealed that An. peditaeniatus, An. hyrcanus, An. sinensis and An. lesteri were likely to undergo hitchhiking or population expansion in accordance with both markers. In addition, the population of an important vivax malaria vector, An. sinensis, has experienced an expansion after a bottleneck in northern and southern Laos. Conclusions The topology of the Hyrcanus group rDNA–ITS2 and mtDNA–COII trees conformed to the morphology-based taxonomy for species classification rather than for that for subgroup division. rDNA–ITS2 is considered to be a more reliable diagnostic tool than mtDNA–COII in terms of investigating the phylogenetic correlation between closely related mosquito species in the Hyrcanus group. Moreover, the population expansion of an important vivax malaria vector, An. sinensis, has underlined a potential risk of malaria transmission in northern and southern Laos. This study contributes to the molecular identification of the Anopheles hyrcanus group in vector surveillance. Graphical abstract

Genetic Diversity of Symbiotic Green Algae of Paramecium bursaria Syngens Originating from Distant Geographical Locations

Paramecium bursaria (Ehrenberg 1831) is a ciliate species living in a symbiotic relationship with green algae. The aim of the study was to identify green algal symbionts of P. bursaria originating from distant geographical locations and to answer the question of whether the occurrence of endosymbiont taxa was correlated with a specific ciliate syngen (sexually separated sibling group). In a comparative analysis, we investigated 43 P. bursaria symbiont strains based on molecular features. Three DNA fragments were sequenced: two from the nuclear genomes—a fragment of the ITS1-5.8S rDNA-ITS2 region and a fragment of the gene encoding large subunit ribosomal RNA (28S rDNA), as well as a fragment of the plastid genome comprising the 3′rpl36-5′infA genes. The analysis of two ribosomal sequences showed the presence of 29 haplotypes (haplotype diversity Hd = 0.98736 for ITS1-5.8S rDNA-ITS2 and Hd = 0.908 for 28S rDNA) in the former two regions, and 36 haplotypes in the 3′rpl36-5′infA gene fragment (Hd = 0.984). The following symbiotic strains were identified: Chlorella vulgaris, Chlorella variabilis, Chlorella sorokiniana and Micractinium conductrix. We rejected the hypotheses concerning (i) the correlation between P. bursaria syngen and symbiotic species, and (ii) the relationship between symbiotic species and geographic distribution.

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams, Ruditapes philippinarum, from Aphae-do (Island), Shinan-gun, Korea

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphaedo (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307-570 (av. 395) μm long and 101-213 (av. 157) μm wide. Most of the daughter sporocysts contained 15-20 furcocercous cercariae each. The cercariae measured 112-146 (av. 134) μm in total length and 35-46 (av. 40) μm in width, with 69-92 (av. 85) μm long body and 39-54 (av. 49) μm long tail. The metacercariae were 210-250 (av. 231) μm in length and 170-195 (av. 185) μm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1-5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8-99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.

First report of Berkeleyomyces basicola causing mango root rot and decline in India

Mango wilt has been a serious constraint in mango (Mangifera indica L.) production in several countries including India (Shukla et al. 2018). Although, several fungal pathogens have been reported associated with the disease, species of Ceratocystis, Verticillium and Lasiodiplodia have been found predominantly responsible for the wilt (Shukla et al. 2018). A twenty-seven-year old mango tree cv. Dashehari at Rehmankhera, Lucknow, Uttar Pradesh, India suffered sudden wilt (Fig. 1A) during February 2020. Though, symptoms were similar to Ceratocystis wilt, no gummosis was observed on trunk or branches which occurred in the majority of Ceratocystis fimbriata infected trees. The infected roots of the wilted tree exhibited dark brown to black discoloration in woody portions (Fig. 1B). Severely affected roots were completely rotten. Similar symptoms of root infection were observed in an additional 16 declining trees within an orchard of 120 trees total (Fig. 2). The infected hard wood samples from live roots of 16 declining and one wilted trees were utilized for isolation by placing stem tissue of discolored and normal colored tissue on surface sterilized fresh carrot discs placed in a moisture chamber (Fig. 1C) for 10 days. Out of 17 tree samples, isolates of Berkeleyomyces basicola (Berk. & Broome) W.J. Nel, Z.W. de Beer, T.A. Duong, M.J. Wingf. (Nel et al. 2018) obtained from 1 wilted and 9 declining trees were transferred to and maintained in pure culture on potato dextrose agar. Isolates were grown for 7 to 10 days at 23±1 °C temperature in the dark. The isolates were characterized by a greyish black compact mycelial colony (Fig. 1D). Two types of spores, endoconidia (phialospores) and chlamydospores (aleuriospores or amylospores) were observed under microscope. The endoconidia were hyaline, cylindrical in shape with 10 to 42 × 3 to 6 μm (n=50) in size (Fig. 1E). Chains of dark colored chlamydospores (3 to 7 spores in chain) of 24 to 52 × 10 to 12 μm (n=50) size were apparent (Fig. 1E&F). Molecular identification of the fungus isolated from the wilted tree was established by amplifying the ITS1-5.8 rDNA-ITS2 region of fungal genomic DNA and the set of ITS primers (ITS 1 and ITS4) (White et al. 1990) followed by sequencing. The sequence has been submitted to the NCBI database vide accession number MT786402. The present isolate (MT786402) shared >99 percent nucleotide similarity with other B. basicola isolates. The phylogenetic tree was constructed using the ITS1-5.8 rDNA-ITS2 sequences of other B. basicola isolates and other Thielaviopsis spp., C. fimbriata, Chalaropsis thielavioides through neighbor joining method using MEGAX software (Fig. 3) (Kumar et al. 2018). The present isolate formed a distinct cluster along with other B. basicola isolates in a separate clade. Koch's postulate was performed under a transparent polycarbonate sheet roof net house at 14.4 and 42.2 °C minimum and maximum temperatures, respectively. A 100 ml macerated culture suspension consisting of 1000 chlamydospores and endoconidia per ml suspension was inoculated in the rhizosphere of mango seedlings planted in sterilized soil filled in earthen pots, using ten replicates for inoculated and uninoculated plants. Symptoms of necrotic root tissue were observed 90 days after inoculation and were consistent with those observed in the field. The same fungus was re-isolated from infected roots and identity was confirmed. All control plants remained symptom-free and B. basicola was not isolated from the roots. Thus, we conclude that B. basicola is capable of causing root rot disease of mango. To the best of our knowledge this is the first report of B. basicola causing mango root rot and decline across the globe, hitherto unreported. The extent of the root necrosis symptoms associated with mature mango trees demonstrates the potential virulence of B. basicola, although its pathogenicity risk on healthy mature trees is still unknown. However, the possibility of severe losses to the mango industry in world number one mango producer country, India cannot be ruled out, if found widespread.

On distinction of the reed species (Phragmites, Poaceae) according to the molecular phylogenetic data

Molecular phylogenetic analysis of the marker sequences ITS and trnL–trnF of the Phragmites australis,P. altissimus, some putative hybrids between them and the sample of P. australis s. l. from Republic of Tuva was performed.ITS1 sequences of the sample from Tuva were studied by the New-generation sequencing method (NGS) revealingintragenomic polymorphism of the ITS sequences in allopolyploid species. We showed good differences betweenP. australis and P. altissimus in the ITS1-5.8S rDNA-ITS2 sequences by many single-nucleotide substitutions anddeletions, in particular, there is a 12 b.p. deletion. P. australis s. l. sample from Tuva (Hemchik river) demonstrates aseparate position on the phylogenetic tree. All reed species studied by us do not have significant differences between eachother according to the chloroplast sequences trnL–trnF.

A new species of Cryptomonas (Cryptophyceae) from the Western Urals (Russia)

A new species, Cryptomonas uralensis Martynenko, Gusev, Kulizin & Guseva sp. nov., is described from western slopes of the Ural Mountains (Russia) based on morphological and molecular data. Phylogenetic relationships inferred from nuclear SSU and LSU rDNA sequences show that the new species forms a clade with C. tetrapyrenoidosa Skuja emend. Hoef-Emden & Melkonian. Comparison of secondary structures of nuclear rDNA ITS2, including analysis of Compensatory Base Changes (CBC), confirms the separation between C. uralensis sp. nov. and C. tetrapyrenoidosa. Cell morphology and sizes of C. uralensis sp. nov. are very similar to C. tetrapyrenoidosa and C. pyrenoidifera, and C. uralensis sp. nov. may thus represent a species that can only be reliably identified using molecular data.

Phylogeny of certain members of Hyrcanus group (Diptera: Culicidae) in China based on mitochondrial genome fragments

Background Species of the Anopheles hyrcanus group are widely distributed in Palearctic and Oriental regions and some of them are important malaria vectors. The cryptic species of An. hyrcanus group was almost impossible to identify based only on their morphology. The phylogenetic relationship of An. hyrcanus group was also not clear. Methods Five members of An. hyrcanus group were identified by rDNA ITS2 sequencing as An. yatsushiroensis, An. belenrae, An. kleini, An. lesteri and An. sineroides. The mitochondrial genome fragments were sequenced and annotated using the mitochondrial genome of An. sinensis as reference. Based on the four segments and Joint Data sequences of these species, and other four anopheline species downloaded from GenBank, intraspecific as well as interspecific genetic distances were calculated and the phylogenetic trees were reconstructed by the methods of neighbor joining, maximum parsimony, minimum evolution and maximum likelihood. Findings Four parts of mitochondrial genomes, which were partial fragments COI + tRNA + COII (F5), ATP6 + COIII(F7 + F8), ND1(F19) and lrRNA (F21), were obtained. All fragments were connected as one sequence (referred as Joint Data), which had a total length of 3393 bp. All fragment sequences were highly conservative within species, with the maximum p distance (0.026) calculated by F19 of An. belenrae. The pairwise interspecific p distance calculated by each fragment showed minor or even no difference among An. sinensis, An. kleini and An. belenrae. However, interspecific p distances calculated by the Joint Data sequence ranged from 0.004 (An. belenrae vs An. kleini) to 0.089 (An. sineroides vs An. minimus), and the p distances of the six members of An. hyrcanus group were all less than 0.029. The phylogenetic tree showed two major clades: all subgenus Anopheles species (including six members of An. hyrcanus group, An. atroparvus and An. quadrimaculatus A) and subgenus Cellia (including An. dirus and An. minimus). The An. hyrcanus group was divided into two clusters as ((An. lesteri, An. sineroides) An. yatsushiroensis) and ((An. belenrae, An. sinensis) An. kleini)). Conclusions The An. hyrcanus group in this study could be divided into two clusters, in one of which An. belenrae, An. sinensis and An. kleini were most closely related. More molecular markers would make greater contribution to phylogenetic analysis.