Neighbor-joining Research Articles

First Report of Xanthomonas hydrangeae Causing Leaf Spot on Oakleaf Hydrangea (Hydrangea quercifolia) in Tuscany (Italy).

Hydrangeas (Hydrangea L.) are popular ornamental plants used in urban landscapes and gardens worldwide for the beauty of their large flowers. In June 2022, dark brown/purple and irregular water-soaked spots coalescing into large areas of necrosis were observed on the leaves of potted Hydrangea quercifolia Bartr. plants growing in two ornamentals nurseries in Pistoia, Tuscany, Italy. Isolations, using two symptomatic plants/nursery, were performed by excising small portions of leaf tissue from the margin of the lesions, and macerating them in 100 μl of sterile distilled water (SDW). After 15 min of incubation, a loopful of the resulting suspension was streaked on yeast extract-dextrose-CaCO3 agar (YDCA) amended with 60 mg L-1 cycloheximide. Mucoid, convex and yellow colonies appeared on YDCA after incubation at 28°C for 48h. After colony purification on yeast extract-nutrient-agar (YNA), two isolates from each nursery were subject to amplification and sequence analysis of the 16S rRNA using universal primers FD1/RD1, for genus identification (Vauterin et al. 2000; Weisburg et al. 1991). All 16S rRNA sequences (OP441051) were identical and BLASTn searches indicated that the isolates belong to the genus Xanthomonas [99.9% nucleotide identity with X. hydrangeae strain LMG 31885 (LR990741.1) and 99.8% with strain LMG 31884T (NR_181958.1)]. For classification at species level, fragments of the housekeeping genes gyrB, rpoD, dnaK, and fyuA, were amplified according to Young et al. (2008). Both strands were sequenced and the consensus sequences aligned using MUSCLE as implemented in MEGA X (Kumar et al. 2018). Homologous sequences were once again identical between the isolates. A neighbor joining phylogenetic analysis of the concatenated fragments, was carried out, using the Tuscan isolate HyQ-Tu1, the type/pathotype strains of the seven pathovars of X.hortorum, proposed by Morinière et al. (2020), the four X.hydrangeae strains characterized by Dia et al. (2021) and the type strain of X.populi as the outgroup. The analysis indicated that HyQ-Tu1 isolate clusters within the X. hydrangeae branch of the recently described X. hortorum - X. hydrangeae species complex (Morinière et al. 2020; Dia et al. 2021; 2022). In agreement with this result, isolates tested positive to the LAMP assay specific for members of the complex's clade C (X. hydrangeae) (Dia et al. 2022). Based on molecular evidence, the isolates were identified as X. hydrangeae (Dia et al. 2021; Oren and Garrity, 2022). Three healthy H. quercifolia potted plants were inoculated by rubbing a 10 µl droplet of a bacterial suspension of X. hydrangeae HyQ-Tu1 adjusted to an OD600 of 0.3 (approx. 108 CFU/ml) in SDW on the adaxial surface of two leaves per plant. Two control leaves/plant were inoculated with SDW. Each inoculated leaf was enclosed for 24h in a polyethylene bag and all plants were maintained in a greenhouse at 28°C. Nine days post inoculation (DPI), leaf spots similar to those observed on naturally infected plants started to become evident on the bacteria-inoculated leaves while control leaves remained asymptomatic throughout the trial (21 DPI). Koch's postulates were fulfilled by re-isolating the bacterium from the symptomatic tissues, obtaining a positive amplification with the clade C-specific LAMP assay (Dia et al. 2022), and confirming that the gyrB sequence was 100% identical to that of X. hydrangeae HyQ-Tu1. Housekeeping gene sequences were submitted to GenBank (OP456006-9). Members of the X. hortorum - X. hydrangeae species complex have been reported to affect H. quercifolia in the USA (Uddin et al. 1996) and H. quercifolia and H. arborescens L. in Belgium (Cottyn et al. 2021). To the best of our knowledge, this is the first documentation of X. hydrangeae causing disease on H. quercifolia in Italy. Further work is required to verify the presence of the bacterium in other European countries and to assess the economic impact that it causes within and outside nurseries.

Barcoding of ornamental freshwater shrimp, Neocaridina denticulata (De Haan, 1844) from the aquatic ornamental market in Purbalingga, Central Java, Indonesia

Abstract. Kusmintarsih ES, Mahmoud HHA, Sastranegara MH, Syakuri H,Nuryanto A, Ambarningrum TB. 2023. Barcoding of ornamental freshwater shrimp, Neocaridina denticulata (De Haan, 1844) from the aquatic ornamental market in Purbalingga, Central Java, Indonesia. Biodiversitas 24: 3601-3608. Indonesia is one of the world’s leading aquatic ornamental shrimps producers and exports. One of the aquatic ornament shrimp species is a Neocaridina denticulate (De Haan, 1844). There is a wide range of colors including red, bright red, yellow, orange, green, blue, violet, black, and color combinations. It is a landlocked Atydiae family species comprising31 species and subspecies. Due to taxonomic difficulties, this species is continually under revision, and the validation of a few species is currently questionable and uncertain for the relationships among the various colors of N. denticulata. The Cytochrome Oxidase gene I (CO1) gene sequences of mitochondrial DNA were used for species DNA barcoding studies. This study aimed to validate the taxonomic status of different colors of N. denticulata species using the mitochondrial DNA or mtDNA Cytochrome oxidase (CO1) gene sequences. Neighbour Joining trees were constructed based on 545 bp of CO1 gene from white, black, red, yellow, blue colors, and their combination. Several sequences were derived from GenBank for relevant species from different countries and the outgroup. The result showed that all samples are homolog, even to N. denticulata from China NC_023823.1.

Weighted ASTRID: fast and accurate species trees from weighted internode distances

BackgroundSpecies tree estimation is a basic step in many biological research projects, but is complicated by the fact that gene trees can differ from the species tree due to processes such as incomplete lineage sorting (ILS), gene duplication and loss (GDL), and horizontal gene transfer (HGT), which can cause different regions within the genome to have different evolutionary histories (i.e., “gene tree heterogeneity”). One approach to estimating species trees in the presence of gene tree heterogeneity resulting from ILS operates by computing trees on each genomic region (i.e., computing “gene trees”) and then using these gene trees to define a matrix of average internode distances, where the internode distance in a tree T between two species x and y is the number of nodes in T between the leaves corresponding to x and y. Given such a matrix, a tree can then be computed using methods such as neighbor joining. Methods such as ASTRID and NJst (which use this basic approach) are provably statistically consistent, very fast (low degree polynomial time) and have had high accuracy under many conditions that makes them competitive with other popular species tree estimation methods. In this study, inspired by the very recent work of weighted ASTRAL, we present weighted ASTRID, a variant of ASTRID that takes the branch uncertainty on the gene trees into account in the internode distance.ResultsOur experimental study evaluating weighted ASTRID typically shows improvements in accuracy compared to the original (unweighted) ASTRID, and shows competitive accuracy against weighted ASTRAL, the state of the art. Our re-implementation of ASTRID also improves the runtime, with marked improvements on large datasets.ConclusionsWeighted ASTRID is a new and very fast method for species tree estimation that typically improves upon ASTRID and has comparable accuracy to weighted ASTRAL, while remaining much faster. Weighted ASTRID is available at https://github.com/RuneBlaze/internode.

Detecting SNP markers discriminating horse breeds by deep learning

The assignment of an individual to the true population of origin using a low-panel of discriminant SNP markers is one of the most important applications of genomic data for practical use. The aim of this study was to evaluate the potential of different Artificial Neural Networks (ANNs) approaches consisting Deep Neural Networks (DNN), Garson and Olden methods for feature selection of informative SNP markers from high-throughput genotyping data, that would be able to trace the true breed of unknown samples. The total of 795 animals from 37 breeds, genotyped by using the Illumina SNP 50k Bead chip were used in the current study and principal component analysis (PCA), log-likelihood ratios (LLR) and Neighbor-Joining (NJ) were applied to assess the performance of different assignment methods. The results revealed that the DNN, Garson, and Olden methods are able to assign individuals to true populations with 4270, 4937, and 7999 SNP markers, respectively. The PCA was used to determine how the animals allocated to the groups using all genotyped markers available on 50k Bead chip and the subset of SNP markers identified with different methods. The results indicated that all SNP panels are able to assign individuals into their true breeds. The success percentage of genetic assignment for different methods assessed by different levels of LLR showed that the success rate of 70% in the analysis was obtained by three methods with the number of markers of 110, 208, and 178 tags for DNN, Garson, and Olden methods, respectively. Also the results showed that DNN performed better than other two approaches by achieving 93% accuracy at the most stringent threshold. Finally, the identified SNPs were successfully used in independent out-group breeds consisting 120 individuals from eight breeds and the results indicated that these markers are able to correctly allocate all unknown samples to true population of origin. Furthermore, the NJ tree of allele-sharing distances on the validation dataset showed that the DNN has a high potential for feature selection. In general, the results of this study indicated that the DNN technique represents an efficient strategy for selecting a reduced pool of highly discriminant markers for assigning individuals to the true population of origin.

Comparison of the evolutionary patterns of DNA repeats in ancient and young invertebrate species flocks of Lake Baikal.

DNA repeat composition of low coverage (0.1-0.5) genomic libraries of four amphipods species endemic to Lake Baikal (East Siberia) and four endemic gastropod species of the fam. Baicaliidae have been compared to each other. In order to do so, a neighbor joining tree was inferred for each quartet of species (amphipods and mollusks) based on the ratio of repeat classes shared in each pair of species. The topology of this tree was compared to the phylogenies inferred for the same species from the concatenated protein-coding mitochondrial nucleotide sequences. In all species analyzed, the fraction of DNA repeats involved circa half of the genome. In relatively more ancient amphipods (most recent common ancestor, MRCA, existed approximately sixty millions years ago), the most abundant were species-specific repeats, while in much younger Baicaliidae (MRCA equal to ca. three millions years) most of the DNA repeats were shared among all four species. If the presence/absence of a repeat is regarded as a separate independent trait, and the ratio of shared to total numbers of repeats in a species pair is used as the measure of distance, the topology of the NJ tree is the same as the quartet phylogeny inferred for the mitogenomes protein coding nucleotide sequences. Meanwhile, in each group of species, a substantial number of repeats were detected pointing to the possibility of non-neutral evolution or a horizontal transfer between species occupying the same biotope. These repeats were shared by non-sister groups while being absent in the sister genomes. On the other hand, in such cases some traits of ecological significance were also shared.

What do molecular data and vibrational communication tell us about the taxonomy of the genus Tyrrhenoleuctra Consiglio, 1957 (Plecoptera: Leuctridae) in Spain?

In this paper we present the results of a study of different populations of the genus Tyrrhenoleuctra, which includes morphologically cryptic species, in the Iberian Peninsula. A total of six populations were analysed from a molecular point of view and a behavioural study was conducted on three of them. Two phylogenetic trees, maximum likelihood (ML) and neighbour joining (NJ), were constructed using both new COI sequences and COI sequences already available in GenBank. Furthermore, intra- and interspecific p-distances were calculated within and between sequences of the different populations. As regards behavioural analyses, the male call of individuals from three of the studied populations was recorded, analysed and described. Both phylogenetic approaches used, ML and NJ, were congruent in discriminating five well-defined clusters, representing the four known Iberian Tyrrhenoleuctra species and a previously unknown taxon. The sequences from the six studied populations produced in this study fell within two clades: T. lusohispanica (four populations) and the new taxon (two populations). These results were supported also by the behavioral study, in which the drumming calls of individuals belonging to the two clades differed in some important parameters such as the number of sequences, intersequence interval and interbeat interval. The obtained results expand the previously known distribution of T. lusohispanica and show the existence of a new species (Tyrrhenoleuctra hynesi sp. n.) within this genus from a more restricted area, that will be described in this paper.

Characterisation of bell pepper (Capsicum annuum L. var. grossum Sendt.) accessions for genetic diversity and population structure based on agro-morphological and microsatellite markers

Bell pepper (Capsicum annuum L. var. grossum Sendt.) is a leading Solanaceous vegetable crop across the world with immense therapeutic and export potential. Such wide cultivation exposes the crop to various biotic and abiotic stresses. Genetic diversity is one of the major prerequisites for any crop breeding programme. Hence, in this study, an attempt was made to evaluate the genetic diversity and population structure of 24 bell pepper genotypes using 25 agro-morphological [9 distinctness, uniformity and stability (DUS) descriptors; 13 quantitative traits; and 3 quality traits] and 82 microsatellite or simple sequence repeat (SSR) markers. Eight DUS descriptors showed polymorphism with maximum morphological variation (H = 1.29) for fruit shape in longitudinal section. Multivariate analysis using Mahalanobis D2 statistic distinguished the genotypes into 11 clusters. With a mean of 4.08 alleles per locus, a total of 151 alleles were generated from 37 polymorphic SSR primers. The polymorphic information content (PIC) ranged from 0.14 (CAMS 451) to 0.76 (CA 525390) with an average of 0.54. The genotypes were divided into two major clusters based on cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) algorithm, whereas the cluster tree using the neighbour-joining (NJ) method identified three major groups. The population was classified into three groups based on structure analysis. On comparing agro-morphological and molecular clusters, 11 genotypes viz., BWT-39, BWT-39-BR, BWT-39-DR, BWT-29, BWT-3Y, BWT-3Y-4L, Kandaghat Selection, BWT-48-AC, BWT-49-AC, EC-464107, and EC-464115, were found common. These genotypes also exhibited considerable resistance to bacterial wilt disease. Mantel's test showed a moderate positive correlation between agro-morphological (DUS descriptors) and molecular data. Therefore, a combined approach of agro-morphological and molecular evaluation can facilitate the selection of diverse parental lines followed by subsequent hybridisation between them to introduce new traits in bell pepper.

Mitogenomic phylogeny reveals the predominance of the Nubian lineage of African wild ass in Indian donkeys

To contribute to the knowledge of maternal genetic diversity in domestic donkeys, this study investigated the mitochondrial DNA variations and analyzed the genetic structure in Indian donkeys based on 31 mitogenome sequences representing four breeds/populations (Agra, Halari, Kachchhi and Spiti). A total of 27 haplotypes with a haplotype diversity value of 0.989 were evident in the donkey genetic resources of India. The genetic differentiation between the investigated populations was evaluated using population pairwise FST values, which showed maximum differentiation between Kachchhi and Halari donkeys. The Neighbor-Joining (NJ) tree based on the whole mitogenome sequence and the Median-Joining (MJ) network for partial D-loop fragment showed clear demarcation of Indian donkeys into Nubian and Somali clades, substantiating African maternal origin of Indian domestic donkeys. The topology of the MJ network excluded the Asian wild asses as the possible progenitors of Indian donkeys. Halari and Agra donkeys showed conformity exclusively to the Nubian lineage of the African wild asses. However, representation of both the Nubian and Somali lineages was observed in Kachchhi and Spiti donkeys. Comprehensive analysis carried out by retrieving D-loop sequences from different countries representing Asia, Africa, Europe and South America revealed existence of shared haplotypes across geographically isolated regions of the globe. This observation is indicative of utility of donkeys as pack animals across inter-continental trading routes during development of human civilizations. Our results represent a valuable contribution to maternal genetic diversity of Indian donkeys and provide insights into the worldwide spread of the species following initial domestication in Africa.

BHLH transcription factor family identification, phylogeny, and its response to abiotic stress in Chenopodium quinoa.

The second-largest transcription factor superfamily in plants is that of the basic helix-loop-helix (bHLH) family, which plays an important complex physiological role in plant growth, tissue development, and environmental adaptation. Systematic research on the Chenopodium quinoa bHLH family will enable a better understanding of this species. Herein, authors used a variety of bioinformatics methods and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) to explore the evolution and function of the 218 CqbHLH genes identified. A total of 218 CqbHLH transcription factor genes were identified in the whole genome, located on 18 chromosomes. A phylogenetic tree was constructed using the CqbHLH and AtbHLH proteins to determine their homology, and the members were divided into 20 subgroups and one unclustered gene. Authors also analyzed 218 CqbHLH genes, conservative motifs, chromosome diffusion, and gene replication. The author constructed one Neighbor-Joining (NJ) tree and a collinearity analysis map of the bHLH family in C. quinoa and six other plant species to study the evolutionary relationship and homology among multiple species. In addition, the expression levels of 20 CqbHLH members from different subgroups in various tissues, different fruit developmental stages, and six abiotic stresses were analyzed. Authors identified 218 CqbHLH genes and studied their biological functions, providing a basis for better understanding and further studying the bHLH family in quinoa.

Comparative genomic analysis of alloherpesviruses: Exploring an available genus/species demarcation proposal and method

The family Alloherpesviridae contains herpesviruses of fish and amphibians. Due to the significant economic losses to aquaculture that herpesviruses can cause, the primary areas of research interest are concerning their pathogenesis and prevention. Despite alloherpesvirus genomic sequences becoming more widely accessible, methods regarding their genus/species classification are still relatively unexplored. In the present study, the phylogenetic relationships between 40 completely sequenced alloherpesviruses were illustrated by the viral proteomic tree (ViPTree), which was divided into three monophyletic groups, namely Cyprinivirus, Ictalurivirus and Batrachovirus. Additionally, average nucleotide identity (ANI) and average amino acid identity (AAI) analyses were performed across all available sequences and clearly displayed species boundaries with the threshold value of ANI/AAI set at 90%. Subsequently, core-pan analysis uncovered 809 orthogroups and 11 core genes shared by all 40 alloherpesvirus genome sequences. For the former, a 15 percent identity depicts a clear genus boundary; for the latter, 8 of them may be qualified for phylogenetic analysis based on amino acid or nucleic acid sequences after being verified using maximum likelihood (ML) or neighbor-joining (NJ) phylogenetic trees. Finally, although the dot plot analysis was valid for the members within Ictalurivirus, it was unsuccessful for Cyprinivirus and Batrachovirus. Taken together, the comparison of individual methodologies provides a wide range of alternatives for alloherpesviruses classification under various circumstances.

First report of Fusarium solani causing wilt disease on Gleditsia sinensis in Tianjin, China.

The Gleditsia sinensis Lam. widely grown in China is a perennial plant with medicinal properties (Zhang et al. 2016). Since 2019, the leaves of G. sinensis have exhibited yellowing and wilting, and the plants have gradually become stunted and dead in Taifeng park of Binhai New Area in Tianjin (39.02° N; 117.65° E). In this park, there are two types of G. sinensis, one is with round branch thorns, the other is with flat branch thorns. The G. sinensis with round branch thorns did not grow well and almost all plants had disease symptoms. The samples were collected on October, 2021 and deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU211018. The disease symptoms consisted of foliage wilt (Figure 1A), plant drying and vascular tissue discoloration (Figure 1B). The stem sections from different plants were surface-disinfested in 0.6% NaClO, wiped with 75% ethanol and rinsed with sterile water. Thirty tissue samples were placed on Potato Dextrose Agar (PDA) medium and cultured at 28℃ for 7 days (Uppala et al. 2013). Thirty fungal isolates with the same morphological characteristics were obtained from the samples. Five representative isolates (PATAU211018-05, PATAU211018-07, PATAU211018-10, PATAU211018-12 and PATAU211018-21) were collected and purified using the single-spore method (Li et al. 2022). Colonies of the five isolates on PDA grew in a circular shape and showed abundant white densely fluffy aerial mycelium (Figure 1C). Morphological characteristics included septate and hyaline hyphae, long cylindrical monophialides (Figure 1D), macroconidia (Figure 1E) and microconidia (Figure 1F). Macroconidia were falcate, 2-5-septate, hyaline, 18-40 × 4-6 μm (n = 50). Microconidia were hyaline, oblong, 0-1-septate, 5-14 × 2-6 μm (n = 50). These morphological characteristics were consistent with the description of Fusarium solani. (Chitrampalam et al. 2018). PATAU211018-12 was randomly chosen for molecular analysis as the representative isolate given the similarity of these isolates. For further identification, the genomic DNA of isolate PATAU211018-12 was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) gene and RNA polymerase II subunit (RPB2) were amplified and sequenced (O'Donnell et al. 2008; Carbone et al. 1999). The sequences of ITS, EF1α, and RPB2 of PATAU211018-12 were deposited in GenBank under the accession no. of OP735578, ON630412 and OP746032, respectively. Phylogenetic trees were constructed in MAGA X (Kumar et al. 2018) using the neighbor-joining (NJ) method based on the concatenated sequences of ITS, EF1α, and RPB2 (Figure 2). The isolate (PATAU211018-12) grouped with F. solani (JS-169) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. solani. Pathogenicity tests were carried out on one-year-old G. sinensis seedlings with round branch thorns (n=6). The F. solani isolate PATAU211018-12 was cultured in Potato Dextrose Broth (PDB) at 28°C on a shaker at 150 rpm for 5 days. Mycelia were filtered through four layers of sterilized lens paper and the conidia were obtained for pathogenicity tests. G. sinensis was infected by F. solani through root soaking method. The roots were inoculated by dipping in conidial suspension with the concentration of 107 conidia/mL for 30 minutes. Control plants (n=6) were treated with distilled water. Plants were in pots indoors at 25℃. At 20 days after inoculation, the leaves of inoculated plants were chlorotic and wilted (Figure 1G), symptoms similar to those observed in the park. In contrast, the leaves of control plants were symptomless (Figure 1H). The pathogenicity assay was repeated three times. The fungal isolate was re-isolated from the disease tissues and verified as F. solani based on morphology and molecular character (ITS, EF1α and RPB2). F. solani has been reported as pathogens on many plants, such as Eriobotrya japonica (Wu et al. 2021), Fragaria × ananassa (Pastrana et al. 2014), Gastrodia elata (Li et al. 2022) and Hedysarum boreale (Uppala et al. 2013). To our knowledge, this is the first report of F. solani causing disease on G. sinensis in China. Identification of F. solani as a disease agent in G. sinensis will assist in disease management for this important tree crop.

First report of Lasiodiplodia theobromae causing stem canker of pomegranate (Punica granatum L.) in India.

In March 2022, cankers and lesions appeared on the branches of 2-3-year-old pomegranate plants grown in four orchards of Hanumangarh, Rajasthan, India. The disease incidence ranged from 5-15%. Field symptoms such as dark brown lesions on one side of the branches, cracked lesions, vascular tissue discoloration and drooping of the plants were noticed. To identify the causative agent, 2 diseased branch samples, showing typical symptoms collected from each orchard 25-30 km apart. The samples were washed with distilled water and small sections of tissue were excised from both symptomatic and asymptomatic areas using a sterile scalpel. Sections were surface sterilized with 1% sodium hypochlorite for 30 sec and 70% ethanol for 2 min followed by rinsing with sterilized water thrice. Sterile sections were dried on sterile filter paper and then transferred onto potato dextrose agar (PDA) amended with streptomycin (100 mgL-1) and incubated at 24±1°C in the dark. Samples (n=5) collected from different orchards produced similar colonies, with greyish white aerial mycelia, which became dark black after 5-7 days. The morphological characteristics of all isolates were observed under microscope. Immature conidia (6.3±1.05*14.7±0.98 μm: average of 50 measurements) were single celled, hyaline, ellipsoid or ovoid, apex rounded and truncated at the base while the matured conidia (8.4±1.41*15.3±1.17 μm: average of 50 measurements) had two cells with dark septa. The conidial morphology of all isolates was in accordance with Lasiodiplodia sp. (Alves et al; 2008) therefore, one representative isolate (HSC-1) was used for molecular identification at species level. Three loci viz., ITS, EF1-a and β tubulin of fungal genomic DNA were PCR amplified using ITS-1/4, EF-F/R and TUB-2A/2B primers, respectively. The amplicons were sequenced and deposited in GenBank, NCBI database with accession no. ON598885 (ITS), ON605203 (EF) and ON605204 (TUB). BLASTn analysis showed similarity with the sequences of Lasiodiplodia theobromae isolates: ITS showed 100% with MK530071.1 (492 bases), EF 99.77% with MT975688.1 (436 bases) and BT 99.76% with MW287586.1 (422 bases). Phylogenetic analysis using Neighbour Joining method revealed close association among L. theobromae isolates. Thus, causative agent associated with stem canker of pomegranate was confirmed as L. theobromae. Further, the same isolate was used for pathogenicity tests on 1-year-old pomegranate plants (n=6). Briefly, 2 cm wound was created in the main stem with a sterile scalpel and a same-size mycelial plug was placed in the wound and wrapped with parafilm. Six plants that were wrapped with uncultured PDA served as control. The inoculated plants were maintained at 26°C and 65-70% RH in a polyhouse. After 4 days parafilm was removed from all plants. The experiment was repeated twice. Inoculated plants produced lesions (0.7 x 5.5 cm; average of 6 measurements) similar to field symptoms after 10-15 days and no such symptoms developed on control plants. The difference between control and inoculated plants was statistically significant (p=0.0001). The fungus was re-isolated from symptomatic tissue and colonies were morphologically similar to HSC-1, thus fulfilling the Koch's postulates. The fungus, L. theobromae causes stem canker and dieback on different host plants and is mainly distributed in tropical and subtropical regions and has been reported on pomegranate from Florida (Xavier et al 2017). To the best of our knowledge, this is the first report of L. theobromae causing stem canker of pomegranate in India.

Morpho-physiological traits and SSR markers-based analysis of relationships and genetic diversity among fodder maize landraces in India.

Maize is an excellent fodder crop due to its high biomass, better palatability, succulency, and nutrition. Studies on morpho-physiological and biochemical characterization of fodder maize are limited. The present study aimed to explore the genetic variation in fodder maize landraces for various morpho-physiological traits and estimation of genetic relationship and population structure. The study on 47 fodder maize landraces revealed significant variation for all morpho-physiological traits except leaf-stem ratio. Plant height, stem girth, leaf-width and number of leaves showed positive correlation with green fodder yield. Morpho-physiological traits-based clustering grouped the landraces into three major clusters, whereas neighbour joining cluster and population structure analysis using 40 SSR markers revealed four and five major groups, respectively. Most landraces of Northern Himalaya-Kashmir and Ludhiana fall into a single group, whereas rest groups mainly had landraces from North-Eastern Himalaya. A total of 101 alleles were generated with mean polymorphic information content value of 0.36 and major allele frequency of 0.68. The pair wise genetic dissimilarity between genotypes ranged from 0.21 to 0.67. Mantel test revealed weak but significant correlation between morphological and molecular distance. Biochemical characterisation of superior landraces revealed significant variation for neutral detergent fibre, acid detergent fibre, cellulose and lignin content. Interestingly, significant, and positive correlation of SPAD with lignin content can be explored to bypass the costly affair of invitro quality assessment for digestibility parameters. The study identified superior landraces and demonstrated the use of molecular markers in genetic diversity assessment and grouping of genotypes for fodder maize improvement.

Species diversity and phylogenetic relationships of snapper (Lutjanidae: Lutjanus) inferred from mitochondrial DNA markers

Snappers (Lutjaniformes: Lutjanidae) are commercially important fishes in tropical and subtropical waters. However, species complexes still exist due to low-level morphological differences. Additionally, current availability of molecular sequences has led to significant changes in fish taxonomy. Therefore, species diversity of lutjanids is still unclear. In this study, we applied two mitochondrial markers (16S rRNA and Cytochrome c oxidase subunit I, COI mtDNA) to investigate species diversity and phylogenetic relationships of lutjanid species collected from the coastal waters of Nghe An – Ha Tinh provinces, Northern Central, Vietnam. A total of 17 Lutjanus species have been identified using morphological and molecular methods. Combined with Genbank sequences, the phylogenetic tree was constructed using Neighbor Joining and Maximum Likelihood approaches based on 16S rRNA and COI mtDNA data set. Two main lineages have been detected with inconsistent basal clades between two topologies. Members of species complexes also showed a certain degree of closely relationships; however, conflicts between two topologies have also been recorded. These data contribute to the assessment of lutjanid biodiversity in Vietnam, and for resource management and conservation.

Genetic Variations of Malaysian And Golden Thai Strains of Climbing Perch and Their Hybrids Based On The Partial Mitochondrial Cytochrome Oxidase Subunit 1 Gene

Rapid genetic improvements from selective breeding are anticipated in many aquaculture species and mitogenome is used to complement the morphological taxonomy of hybrids to evaluate its genetic structures. A study of the genetic variations within the two strains (Malaysian strain and Golden Thai strain) of Anabas testudineus (Bloch, 1792) and their hybrids, phylogenetic trees based on cytochrome oxidase subunit 1 (CO1) of the partial mitochondrial DNA gene were constructed using Maximum Likelihood and Neighbor Joining approaches. The findings support the monophyletic status of the genus with only one haplogroup from which other haplotypes were descended and of a single common ancestor. The individual fishes’ phylogenetic relationships revealed two major clades and Saurida undosquamis as an outgroup. All the groups had high haplotype diversity, except for the hybrid Malaysian strain × golden Thai strain (0.1540±0.126). This suggests that different fish species in each of these studies had different nucleotide compositions in their mitochondrial genomes. The highest number of haplotypes and the presence of distinct haplotypes in the Golden Thai strain × Malaysian strain hybrid point to the absence of recent or regular gene flow as well as high genetic diversity within the hybrids. This study demonstrated that the mtDNA diversity of Anabas testudineus from Malaysia and Thailand had been preserved. Studies on the population genetic diversity and molecular evolution of anabas fish species can benefit from the data provided by this work.

Genetic polymorphism and variability in the Guangdong Hakka, Teochew, and Cantonese groups: A comprehensive analysis of 19 X-STRs.

X chromosomeshort tandem repeat (X-STR) loci are playing an increasingly important role inforensic work, identifying female traces in male contamination and explainingcomplex kinship analyses. In this study, we analyzed thegenetic polymorphism of 19 X-STR loci in the Guangdong Hakka, Teochew and Cantonese groups, respectively, aswell as in the Guangdong Hakka, Teochew andCantonese pooled Han. The genetic diversity and forensic characteristics of the19 X-STRs and 7 linkage groups were investigated, respectively. The experiments showed that the genetic diversity (GD) and polymorphism information content (PIC) in the pooledGuangdong Han ranged from 0.5320 to 0.9234 and 0.4369 to 0.9171, respectively, and the cumulative power of discrimination for males (PDM), power of discrimination for females (PDF) and mean paternity exclusion chance (MEC) were higher than 0.9999999, indicating that the 19 X-STRs had high geneticpolymorphism and discriminatory power. Genetic differences among Chinese Hansubgroups and among different Chinese populations were investigated byphylogenetic reconstruction and principal component analysis (PCA), respectively. Genetic analyses based on neighbor-joining (NJ) tree and principal component analysis plot showed that Cantonese, Teochew and Hakka were closely genetically related, and different populations with closer linguistic components had more genetic affinity. This study adds to the forensic X-STR database and demonstrates the forensic efficiency of 19 X-STRs for the Hakka, Teochewand Cantonese populations in Guangdong, and the pooled Han of Hakka, Teochewand Cantonese people in Guangdong.

DNA Barcoding and Phylogenetics Relationship of Pangasiid Catfishes in Peninsular Malaysia Revealed the Impacts of Aquaculture on the Native Species Conservation

Pangasiids are an economically significant group of catfish, and many pangasiids are threatened in the wild from anthropogenic pressures, including increases in fishing pressure, habitat degradation, and improperly managed aquaculture practices. This study demonstrates the usage of DNA barcoding of the Cytochrome Oxidase subunit I (COI) gene as an identification tool in detecting potentially threatening invasive pangasiid species by establishing the diversity and phylogenetic relationship of Pangasiidae catfishes in Peninsular Malaysia. A neighbour-joining (NJ) dendrogram (Kimura-2-parameter model) generated five clades to represent distinct genera. Pangasius was further subdivided into two clades (Clade A: Pangasius bocourti-P. djambal and Clade B: P. nasutus-P. conchophilus). Given the marginal genetic divergence, indigenous and non-native species should be treated cautiously in allopatrically distributed species. The analysis used Automatic Barcode Gap Discovery (ABGD) and revealed barcode gaps between the intraspecific and interspecific distances. The sequences were partitioned into five groupings, corresponding with the species delineation based on the distribution of pairwise differences, which could not be differentiated using the NJ dendrogram. ABGD allows the recognition of one or two additional species using the recursive approach, but other taxonomic methods should be considered for a solid conclusion. DNA barcoding demonstrates the identification of closely related species, thus justifying its application towards the conservation of these fish.

First Report of Nigrospora aurantiaca Causing Leaf Blight Disease of Peanut in China.

In June 2021, a previously unreported leaf blight disease of peanut (Arachis hypogaea) was observed on field-grown peanut (Jinhua19) in Laixi city, Shandong province of China. Approximately 5% of plants showed disease symptoms in the fields we investigated. The symptoms first appeared as yellow round or irregular spots on leaves, and then the spots became brown. As the disease progressed, spots became larger and even converge, which later produced leaf chlorosis and abscission. Symptomatic leaves were cut into small pieces, surface disinfested with 70% ethanol for 30s, 1% NaClO for 60s, rinsed three times in sterile water, dried on sterile filter papers, placed on potato dextrose agar (PDA) media, and incubated at 25°C in darkness. Fungal cultures were initially white, with red pigment, then turned gray, and eventually turned black, and aerial hyphae were dense. Conidia were spherical or slightly ellipsoidal, black, smooth, and 8.6 to 11.5 × 8.7 to 14.5μm (n=50). Morphological characteristics of the isolates matched the description of Nigrospora aurantiaca (Wang et al. 2017). Molecular identification was performed by sequencing beta tubulin gene (TUB) with Bt2a/Bt2b and translation elongation factor 1-alpha (TEF) with EF1-728F/EF1-986R (Wang et al. 2021) of a representative isolate ZHX11. TUB (OK489789) and TEF (OK489790) of ZHX11 obtained 100% (401/401 nucleotides) and 99.64% (279/279 nucleotides) similar to those of N. aurantiaca (MN329935, MN264010), respectively. Alignment was conducted separately for each gene set using Clustal W algorithm implemented in MEGA 7.0 (Kumar et al. 2016), and multi-gene (TUB and TEF) phylogenetic analyses using Neighbor-Joining (NJ) method showed that the isolate was N. aurantiaca. To complete Koch's postulates, nine 2-week-old peanut (Zhonghua 12) seedlings were sprayed with conidia suspensions (106 conidia mL-1 in 0.05% Tween 20 buffer). The same number of seedlings were only treated with 0.05% Tween buffer as controls. The experiment was repeated three times. Plants were incubated in a growth chamber (30°C in the day and 25°C at night, a 12-h photoperiod and 80% RH). Ten days after inoculation, typical symptoms were observed on inoculated leaves but not on the controls. N. aurantiaca was reisolated from the diseased leaves but not from the controls. N. sphaerica was observed on peanut in China (Liu et al. 2020). To our knowledge, this is the first report of N. aurantiaca causing leaf blight on peanut in shandong province, China. These findings will help to develop better preventive measures in accordance with the emergence of the new disease.

Morphological and molecular characteristics of Donax incarnatus (Bivalvia: Donacidae) from Badur Beach, Madura Island, Indonesia based on the COX1 gene

Abstract. Wijaya CB, Ambarwati R, Rahayu DA. 2023. Morphological and molecular characteristics of Donax incarnatus (Bivalvia: Donacidae) from Badur Beach, Madura Island, Indonesia based on the COX1 gene. Biodiversitas 24: 2805-2813. Donacidae, a family of marine bivalve mollusks, has a high diversity of morphological traits, including shape and color pattern variations. However, these variations often led to errors in morphological identification, necessitating the use of molecular identification methods for accurate species identification and support. This study aims to analyze the morphological and molecular characteristics of Donax incarnatus Gmelin, 1791 collected from Badur Beach of Madura Island, Sumenep District, East Java Province, Indonesia, using the COX1 gene as a molecular marker. Morphological characterization was conducted through qualitative and quantitative observations, while sequencing results were analyzed using bioinformatics software. The results showed that the exterior color of D. incarnatus has three morphological variations, namely purple-black (OQ692130.1), yellowish orange (OQ692131.1), and white (OQ692132.1). Molecular characterization indicated that the similarity value of D. incarnatus with these variations ranged from 96.06% to 99.61%. At the same time, the alignment results of all samples with reference species and outgroups obtained five variations of nucleotide bases. The average genetic distance between D. incarnatus and the in group was 0.01. The phylogenetic tree results obtained using Neighbor-Joining (NJ) and Maximum Likelihood (ML)methods were categorized into two subclusters: D. incarnatus species from Thailand and the study sample from Badur Beach, Madura Island, Indonesia, were placed in the same cluster.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Cinnamomum camphora in China.

Camphor tree (Cinnamomum camphora) has a wide distribution in the world and is mainly distributed in the South and southwest in China. It can be used as both a wood and a medicine, with high value in industry, medicine, and ecology. In May 2022, it was observed that the approximately ten to fifteen years old Camphor trees were exhibiting witches'-broom, small leaf morphology and chlorosis, and leaf drop in Panzhihua City, Sichuan Province, China. The witches'-broom symptoms consisted of many small branches with little leaves at the top of branches. It was named C. camphora witches'-broom disease (CCWB) and was found in some areas of Miyi, Yanbian, Renhe Xiqu and Dongqu counties. More than 28% of the plants were infected on the five areas surveyed. Total 100 samples were collected from five areas, with 15 symptomatic plants and 5 asymptomatic plants each area. The lateral stem tissues were observed under a scanning electron microscope (Hitachi S-3000N). The nearly spherical bodies were found in the phloem sieve cells of symptomatic plants. Total DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), ddH2O was used as the negative control, and Dodonaea viscose witches'-broom disease plants were used as the positive control. The nested PCR was employed to amplify the 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and PCR amplicon of 1.2 kb were amplified (GenBank accessions OP662614; OP662615; OP662616). The direct PCR specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb with primer pair rp(I)F1A and rp(I)R1A (Lee et al. 2003) (GenBank accessions OP649592; OP649593; OP649594). The fragment from 25 symptomatic samples was consistent with the positive control, and asymptomatic plants were negative, confirming an association of a phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of CCWB phytoplasma showed that it has a 99.44% similarity with Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.59% identity with rapeseed phyllody phytoplasma (GenBank accession CP055264). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the query 16S rDNA fragment of CCWB phytoplasma is most similar to the reference pattern of the 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The phytoplasma is identified as 'Ca. Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the CCWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In addition, the plants were positive for the phytoplasma using nested PCR after grafting for 30 d in nursery conditions. It is noteworthy that the plants were seriously damaged by aphid, Psyllidae and Ceroplastes. It is speculated that the insects of Homoptera typically transmit phytoplasmas by feeding on plant sap, thus it is necessary to control aphids in order to control the C. camphora witches'-broom disease. To the best of our knowledge, Camphor tree is a new host plant of 'Ca. Phytoplasma asteris' in China. The newly emerged disease is a threat to Camphor tree production.