First observation on morphological and molecular characters of Bitylenchus ventrosignatus (Tobar Jiménez, 1969) Siddiqi, 1986 isolated from tomato in Dalmada, South Africa

Phylogeny of Tunicata inferred from molecular and morphological characters

Kiwi is becoming one of the most important fruit in subtropical regions of South Africa with altitudes that confer sufficient chilling requirements. During a survey on biodiversity of plant-parasitic nematodes of kiwi in Magoebaskloof in Limpopo Province, several plant-parasitic nematodes were discovered, with Meloidogyne species occurring at the highest frequency. Nematodes were sampled from roots and the rhizosphere of one stunted Kiwi tree, extracted using the tray method and then fixed. The morphological characters fit well with those of M. hapla. The molecular approach using ITS and 28S rDNA, along with the related phylogenetic analysis, placed the examined population in a group with other populations of M. hapla. Kiwi is being reported as a new host for M. hapla in South Africa.

Plant-parasitic nematodes are the main nematodes that live in the soil, causing yield loss in various crops. As endoparasites, Pratylenchus species can reduce the yield of grass and other plants by feeding on their roots. Because of its parasitic nature on plants, this species requires careful identification; in South Africa's Limpopo Province, grasslands are among the most vital agricultural resources. Therefore, this molecular study was conducted in 2023 at Limpopo University to identify the nematode from South Africa's soils using 28S rDNA marker. The recovered nematode was extracted using the tray method, and then its DNA was extracted using the chelex method. The nematode was identified as Pratylenchus. Afterward, 28S rDNA was amplified using specific primers to identify the nematode at the molecular level. The Nblast analysis based on the large subunit ribosomal DNA showed South African Pratylenchus had 99% similarity (OQ343703) with the South African population. Phylogenetic analysis using maximum likelihood placed this species with those molecularly identified as Pratylenchus in the same clade with highly supported (100) bootstrap values. In conclusion, this species was identified using 28S rDNA; however, other rDNA markers, such as ITS rDNA and mtDNA is recommended for a better understanding of Pratylenchus phylogeny.

During a survey of plant-parasitic nematodes in South Africa's Limpopo Province, two species of Helicotylenchus were identified, namely H. dihystera and H. pseudorobustus. The morphological and molecular characteristics of these species were found to be consistent with those of the known species. A phylogenetic analysis of Helicotylenchus populations based on 28S rDNA was conducted, and it was found that the H. dihystera identified in this study belonged to the same group as other H. dihystera specimens with a 1.00 posterior probability support. Moreover, phylogenetic analysis of H. pseudorobustus based on 18S rDNA placed the test population close to other H. pseudorobustus specimens with 0.97 posterior probability. Scanning electron microscopy (SEM) for Helicotylenchus species also revealed noticeable dissimilarities in the labial disc and lateral field of the tail region between the two species from the present study, including H. pseudorobustus, and H. dihystera. The redundancy analysis (RDA) showed that H. pseudorobustus had a correlation with pH and clay of the soil. In conclusion, despite the challenges associated with identifying Helicotylenchus species, SEM and rDNA markers can be considered as highly effective tools to distinguish the species correctly and accurately.

During a survey on the biodiversity of plant-parasitic nematodes of natural areas in Botswana, Mesocriconema sphaerocephalum was discovered around the rhizosphere of the wild grass. The nematodes were extracted using the tray method and then fixed according to the available protocols. The morphological characters fit well with the M. sphaerocephalum. Besides, molecular aspects using 18S and 28S rDNA were studied. The phylogenetic analysis of 18S and 28S rDNA placed the examined population with other populations of M. sphaerocephalum in a group. According to the knowledge, this is the first report of M. spaherocephalum from Botswana.

Predatory nematodes are the main nematodes that live in the soil assisting with the biological control of the plant parasitic nematodes associated with various crops. Mononchus species is a predatory nematode and therefore can feed on the other nematodes that exist in the soil. This species is important due to its critical role in the biocontrol strategy and therefore must be identified correctly. The soil samples were collected from a tomato field in Limpopo Province in South Africa. This molecular study was conducted in 2022 at Limpopo University to identify the nematode species from South Africa's soils using 28S rDNA marker. The recovered nematode was extracted using the tray method, and then its DNA was extracted using the chelex method. The nematode was identified as Mononchus sp. Afterwards, 28S rDNA was amplified using specific primers to identify the nematode at the molecular level. The Nblast analysis based on the large subunit ribosomal DNA showed South African Mononchus sp. had 99 % similarity (KY750781) with a Mexican population. Phylogenetic analysis using maximum likelihood placed this species with those molecularly identified as Mononchus in the same clade with highly supported (100) bootstrap values. In conclusion, this is the first 28S rDNA of a Mononchus from South Africa. Additionally, though this species was identified using 28S rDNA however, the use of other rDNA markers such as ITS rDNA and 18S rDNA for a better understanding of Mononchus phylogeny is recommended.

Plant-parasitic nematodes are the main nematodes that live in the soil, causing yield loss in various crops. Since Xiphinema oxycaudatum is an ectoparasite, it can reduce crop yields by feeding on the roots of crops like maize. Correct identification of this species is crucial as maize is a vital crop in South Africa's Limpopo Province. Therefore, this molecular study was conducted in 2023 at Limpopo University to identify the nematode from South Africa's soils using 28S rDNA marker. The recovered nematode was extracted using the tray method, and then its DNA was extracted using the chelex method. The nematode was identified as Xiphinema. Afterward, 28S rDNA was amplified using specific primers to identify the nematode at the molecular level. The Nblast analysis based on the large subunit ribosomal DNA showed South African X. oxycaudatum had 99% similarity (MK966417) with the South African population. Phylogenetic analysis using maximum likelihood placed this species with those molecularly identified as X. oxycaudatum in the same clade with highly supported (100) bootstrap values. While 28S rDNA was used to successfully identify this species, further investigation into the phylogeny of X. oxycaudatum will benefit from the addition of other rDNA markers such as ITS rDNA and mitochondrial DNA.

Dorylaimida members are medium to large nematodes that live in the soil and create a balanced environment towards improving yield in various crops. Aporcella species are omnivores and, therefore, can feed on other nematodes around the roots of maize, resulting in better production. This species must be identified correctly because it is a beneficial nematode and can enhance the yield of maize through the microbial balancing of the soil. On the other hand, maize is one of the most important crops in Limpopo Province in South Africa. Therefore, this molecular study was conducted in 2023 at Limpopo University to identify the nematode from South Africa's soils using 28S rDNA marker. The recovered nematode was extracted using the tray method, and then its DNA was extracted using the Chelex method. The nematode was identified as Aporcella simplex (Thorne and Swanger). Afterwards, 28S rDNA was amplified using specific primers to identify the nematode at the molecular level. The Nblast analysis based on the large subunit ribosomal DNA showed that South African A. simplex had 99% similarity (MN262455) with the South African population. Phylogenetic analysis using maximum likelihood placed this species with those molecularly identified as A. simplex in the same clade with highly supported (100) bootstrap values. In conclusion, this species was determined using 28S rDNA; however, other rDNA markers, such as ITS rDNA and mtDNA, are recommended for a better understanding of A. simplex phylogeny.

Panagrolaimidae members are bacterivorous with global distribution. There is no information on this species' morphology and molecular characterization in South Africa. During a survey of Molepo dam, Limpopo Province, a population of Propanagrolaimus nematodes belonging to the family Panagrolaimidae was recovered and determined utilizing conventional and molecular characters. Propanagrolaimus siweyae sp. n., was confirmed by morphological and molecular informations. Propanagrolaimus siweyae sp. n. is characterized by a body length 1026–1258 µm (a = 39.4–45.5, b = 5.4–6.5, c = 8.1–10.0, c’ = 6.9–7.8, V = 53–57), post vulval uterine sac 25–53 µm, and tail length 108–158 µm long. The scan electron microscopy (SEM) photographs revealed a tessellated cuticle and six slightly separated lips. The discriminant analysis placed populations of P. siweyae sp. n. close to P. hygrophilus with clear separation from the mentioned species. Partial sequences of the 18S and 28S regions of the ribosomal DNA gene were amplified for P. siweyae sp. n. The phylogenetic analysis grouped P. siweyae sp. n. in a clade with 1.00 and 0.87 posterior probability values together with other Propanagrolaimus and Halicephalobus based on 18S and 28S rDNA, respectively. Principal component analysis revealed no correlation between Propanagrolaimus and water parameters. However, the result showed a high correlation with diatoms (r = 0.937) in Molepo dam (site MD-6). In contrast, The PCA showed salinity, pH, and temperature with no significant effect on nematodes in Molepo dam, Limpopo Province, South Africa. In conclusion, the usefulness of free-living nematodes as bioindicators is an advantage to studying pollution in aquatic systems. Limpopo Province is facing pollution in the river and dam systems. Therefore, finding a bioindicator is critical to examine the ecological role of free-living nematodes such as Propanagrolaimus.

Tomato is one of the premier vegetables in the world and is widely cultivated for higher yield and economic importance; thus, the area under cultivation is increasing. Pandoraea species can be a risky pathogenic bacterium to tomato plants, impacting the yield. They are gram-negative, motile, and rod-shaped. In 2022, the University of Limpopo conducted a study to identify and name the Pandoraea bacterium found in a tomato field located in Limpopo Province, South Africa. Molecular methods were employed, and the Chelex approach was utilized to extract the 16S rDNA marker, enabling the identification of bacteria present in the soil of tomato fields in South Africa. After conducting tests using specific primers to amplify the 16S rDNA, we have identified the bacterium as Pandoraea. Our analysis indicates that the strain of Pandoraea found in South Africa shares a 90% similarity with a population from Belgium (LR536859). Furthermore, maximum likelihood phylogenetic analysis indicates that this species is grouped with other Pandoraea species, with highly supported (100) bootstrap values. While the 16S rDNA method successfully identified this species, we recommend utilizing other DNA markers for a more comprehensive understanding of the Pandoraea phylogeny.

Pseudomonas is a genus of Gram-negative, Gammaproteobacteria, belonging to the family Pseudomonadaceae. Pseudomonas cichorii is a Gram-negative soil bacterium that is pathogenic to plants. It has a wide host range and can have an important economic impact on lettuce, celery, and chrysanthemum crops. Pseudomonas cichorii can be a risky pathogenic bactererium in vegetables, including spinach, and therefore have an effect on the fresh production of spinach. This study was conducted to isolate and identify P. cichorii from a spinach field in Limpopo Province, South Africa. The molecular study was conducted in 2023 at Limpopo University to identify the bacterium from South Africa's soils using 16S rDNA marker. The DNA was extracted using the Chelex method. The bacterium was identified as P. cichorii. The Nblast analysis showed South African P. cichorii had 99% similarity with a population from South Africa (FJ477103). Phylogenetic analysis using maximum likelihood placed this species with those molecularly identified as P. cichorii in the same clade with highly supported (100) bootstrap values. The species was identified using 16S rDNA however, the use of other DNA markers can be evaluated for a better understanding of P. cichorii phylogeny.

Streptomyces species are gram-positive and are the largest genus of Actinomycetota and the type genus of the family Streptomycetaceae. Over 500 species of Streptomyces bacteria have been described. Streptomyces includes several species that can be used as a biocontrol agent for managing plant pathogenic bacteria and fungi threatening the crops. Therefore, they become important bacteria for improving yields in potato production. This study was conducted to isolate and identify Streptomyces from a potato field in Limpopo Province, South Africa. Therefore, this molecular study was conducted in 2023 at Limpopo University to identify the bacterium associated with potato filed from South Africa's soils using 16S rDNA marker. The DNA was extracted using the Chelex method. The bacterium was identified as Streptomyces. The Nblast analysis showed South African Streptomyces has 99% similarity with a population from South Africa (HQ823600). Phylogenetic analysis using maximum likelihood placed this species with those molecularly identified as Streptomyces in the same clade with highly supported (100) bootstrap values. In conclusion, this species was identified using 16S rDNA properly. However, using other DNA markers for a better understanding of Streptomyces phylogeny is recommended.

Enterobacter species are gram-negative, motile, non-spore-forming, rod-shaped, facultatively anaerobic bacteria of the Burkholderiaceae family. Burkholderia includes several species, which can be risky pathogenic bacteria to vegetables, including cabbage, and therefore have an impact on the fresh production of cabbage. This study was conducted to isolate and identify Burkholderia from a cabbage field in Limpopo Province, South Africa. Therefore, this molecular study was conducted in 2023 at Limpopo University to identify the bacterium associated with cabbage filed from South Africa's soils using 16S rDNA marker. The DNA was extracted using the Chelex method. The bacterium was identified as Burkholderia. The Nblast analysis showed that South African Burkholderia had 95% similarity with a population from South Africa (MG593873). Phylogenetic analysis using maximum likelihood placed this species with those molecularly identified as Burkholderia in the same clade with highly supported (100) bootstrap values. In conclusion, this species was identified using 16S rDNA properly. However, using other DNA markers for a better understanding of Burkholderia phylogeny is recommended.

South Africa is a significant producer and exporter of avocados in the world. Xylosandrus crassiusculushas was found on mainland Africa for many years but the present study represents the first record of bark beetle in South Africa in Limpopo Province. Its presence infesting important agronomic tree crops such as avocado suggests that it requires further study. Therefore, this molecular study was conducted in 2022 at the University of Limpopo to identify the bark beetle in the South African avocado orchard. The recovered bark beetle was isolated from the bark of avocado in Magoebaskloof region in Limpopo Province. Then its DNA was extracted using the Chelex method, and the phylogenetic position of the beetle was studied using 28S rDNA. The Nblast analysis based on the large subunit ribosomal DNA showed 99% similarity with a population of Xylosandrus crassiusculus from South Africa (MT229960), USA (GU808593), and Taiwan (MT120970). In addition, phylogenetic analysis using the maximum likelihood method as implemented in mega x software was done. The phylogenetic analysis put the South African populations of X. crassiusculus together with other X. crassiusculus with a 100-bootstrap value. In conclusion, the results showed that 28S could detect the X. crassiusculus, and was verified by the molecular method.

South Africa is a significant producer and exporter of avocados in the world. Xylosandrus crassiusculushas was found on mainland Africa for many years but the present study represents the first record of bark beetle in South Africa in Limpopo Province. Its presence infesting important agronomic tree crops such as avocado suggests that it requires further study. Therefore, this molecular study was conducted in 2022 at the University of Limpopo to identify the bark beetle in the South African avocado orchard. The recovered bark beetle was isolated from the bark of avocado in Magoebaskloof region in Limpopo Province. Then its DNA was extracted using the Chelex method, and the phylogenetic position of the beetle was studied using 28S rDNA. The Nblast analysis based on the large subunit ribosomal DNA showed 99% similarity with a population of Xylosandrus crassiusculus from South Africa (MT229960), USA (GU808593), and Taiwan (MT120970). In addition, phylogenetic analysis using the maximum likelihood method as implemented in mega x software was done. The phylogenetic analysis put the South African populations of X. crassiusculus together with other X. crassiusculus with a 100-bootstrap value. In conclusion, the results showed that 28S could detect the X. crassiusculus, and was verified by the molecular method.