Transcriptomics Analysis and Candidate Genes Associated With Xinjiang Jujubes in Response to Alternaria Alternata Infection

BackgroundBlack spot disease, caused by Alternaria altrenata, is one of the most destructive diseases of jujube worldwide. To better understand the resistance mechanisms of jujube to A. altrenata infection to be able to improve disease control and resistance breeding. Two different cultivars, Zizyphus jujuba Mill. var. Jun jujube (susceptible) and Zizyphus jujuba Mill. var. Hui jujube (resistant), were tested. ResultsIn this study, we identified 2235 differentially expressed genes (DEGs) in the disease-resistant cultivar and 4958 in the susceptible cultivar. To better understand these DEGs, the datasets were analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genome (KEGG) database. Most of them were associated with plant phytohormone synthesis and signal transduction, flavonoid synthesis, and glutathione metabolism. The expression of 6 DEGs associated with disease resistance were detected by real time-quantitative polymerase chain reaction (RT-qPCR), consistent with the results of Illumina transcriptome sequencing. Moreover, the expression level of the six DEGs differently in Jun jujube and Hui jujube, verified they are defense response factors. ConclusionsThe present study identified several candidate resistance genes and signal transduction pathways that may contribute to black spot disease resistance in jujube, which will assist the investigation of resistance mechanisms in the response of jujube to A. altrenata infection.

TINGGINYA ANGKA KECELAKANAAN AKIBAT PEMBANGUNAN JALUR 3 DI KOTA BANGKO

Masalah kecelakaan lalu lintas pada jalur tiga dikota Bangko selama kurun waktu 2017-2020. Tujuan penelitian ini adalah Mengetahui penyebab terjadinya kecelakaan lalu lintas pada jalur tiga dikota Bangko dan Menentukan black Spot. Penelitian ini adalah penelitian deskriptif retrospektif data sekunder dari laporan kecelakaan lalu lintas pada jalur tiga dikota Bangko dan data primer didapat langsung kelapangan. Empat faktor yaitu, faktor manusia sebanyak 134 kasus, faktor sarana sebanyak 66 kasus, faktor prasarana sebanyak 18 kasus dan faktor cuaca sebanyak 10 kasus, terdapat beberapa keselahan yang menjadi salah satu faktor tingginya angka kecelakaan. Rambu-rambu lalu lintas yang tidak ada, peletakan bukaan median jalan yang tidak tepat. Analisis EAN dengan nilai BKA dan UCL dimana dari 5 segmen terdapat 3 segmen/Black Site yang merupakan daerah rawan, yaitu KM 0 + 700 - KM 1 + 400 = 1248, KM 2 + 100 - KM 2 + 800 = 723 dan KM 2 + 800 - KM 3 + 500 = 588, terdapat Black Site yang memiliki nilai paling tinggi diantara lainnya, yaitu Black Spotnya terletak pada segmen km 0 + 700 – km 1 + 400 = 1248. Hasil analisa bahwa tingkat kecelakkan (Rate Of Accident) tertinggi terjadi pada tahun 2020.

ANTI-TYROSINASE AND ANTI-OXIDANT POTENTIAL OF METHANOLIC EXTRACTS OF SELECTED CITRUS BERGAMIA AND FICUS CARICA PARTS

Tyrosinase is a key enzyme in melanogenesis and its high activity leads to increasedpigmentations causing skin disorder like freckles, melanosoma and black spot. Therefore to search for new tyrosinase inhibitors is desirable. In present study, methanolic (MeOH) extracts from leaves, fruit peel and pulp of Citrus bergamia (CB) and, leaves and fruitof Ficus carica (FC) were prepared which were further process for fractional ethyl alcohol (EA), n-hexane (n-Hx) and chloroform (CHCl3)extractions (total 20 extracts) aiming to test their anti-tyrosinase potential, in-vitro. Our results confirmed that all MeOH FC and CB extracts showed significant anti-oxidant activity with IC50 range of 461.9 ± 16.1µg/ml to 2324.4 ± 116.1 µg/ml. Moreover, CB and FC all 20 extracts have significant anti-tyrosinase activity with IC50 range of 13.9 ± 0.5 µg/ml to 320.5 ± 3.3 µg/ml. Interestingly, CB MeOH-EA peel and leaf extracts showed tyrosinase inhibition (IC50) 13.9 ± 0.5 µg/ml and 17.2 ± 0.8 µg/ml, respectively) is better than all other tested extracts and positive control kojic acid (IC50=18.75±5.29µg/ml). Thus, CB MeOH-EA peel extract with lowest IC50 value among all the tested extracts and kojic acid is proposed as potent candidate to control tyrosinase rooted hyperpigmentation.

Pathogenic Fungi Diversity of ‘CuiXiang’ Kiwifruit Black Spot Disease during Storage

Kiwifruit black spot disease has become increasingly widespread in many ‘CuiXiang’ kiwifruit plantings regions. This research was aimed at the pathogenic microorganisms of black spot of the ‘CuiXiang’ cultivar. Physiological, morphological and transcriptional characteristics between black spot fruit and healthy fruits were evaluated. Then, it applied a high-throughput internal transcribed spacer (ITS) sequencing to analyze the black spot disease microbial community. The cell structure showed that mycelium was attached to the surface of the kiwifruit through black spot, and that consequently the mitochondria were damaged, starch particles were reduced, and shelf life was shortened. Transcriptome revealed that different genes in kiwifruit with black spot disease were involved in cell wall modification, pathogen perception, and signal transduction. ITS sequencing results described the disease-causing fungi and found that the microbial diversity of black spot-diseased fruit was lower than that of healthy fruit. We predict that candidate pathogenic fungi Cladosporium cladosporioides, Diaporthe phaseolorum, Alternaria alternata, and Trichothecium roseum may cause black spot. This study was to explore the pathogenic fungal community of ‘CuiXiang’ kiwifruit black spot disease and to provide essential information for field prevention.

The Effects of Serendipita indica and Guanidine-Modified Nanomaterial on Growth and Development of Cabbage Seedlings and Black Spot Infestation

To ensure sustainable agricultural production and protection of crops from various biotic and abiotic stresses, while keeping in view environmental protection, by minimal usage of chemicals, the exploitation of beneficial microorganisms and modern nanotechnologies in the field of agriculture is of paramount importance. This study aimed to investigate the effects of Serendipita indica and guanidine-modified nanomaterial on the growth, and other selected parameters, of cabbage, as well as incidence of black spot disease. S. indica was applied in substrate and by seed inoculation. S. indica had a positive impact on the development of plants, and resulted in reduced black spot severity. The maximum plant height (119 mm) and number of leaves (8.3) were observed in S. indica-treated plants. Pigments were enhanced, i.e., chlorophyll a (0.79 mg/g), chlorophyll b (0.22 mg/g), and carotenoid content (0.79 mg/g), by substrate treatment. The highest antioxidant capacity (9.5 mM/L), chlorophyll a and b (1.8 and 0.6 mg/g), and carotenoid content (1.8 mg/L) were reported in S. indica seed treatment. S. indica treatment resulted in 59% and 41% disease incidence decrease in substrate and seed treatment, respectively. Guanidine-modified nanomaterial was seen to be effective in improving plant growth and reducing disease incidence; however, it did not perform better than S. indica. Application of nanoparticles resulted in enhanced normalized difference vegetation index and fluorescence by increasing chlorophyll a, b, and carotenoid content. Nitrogen content was the highest in plants treated with nanoparticles. However, the effect of the combined application of fungus and nanoparticles was similar to that of S. indica alone in substrate treatment, although negative impacts were reported in the biochemical parameters of cabbage. S. indica has great potential to enhance plant growth and manage Alternaria incidence in cabbage crops.

First report of Stemphylium eturmiunum causing leaf spot and black spot on apple in Zhaotong, Yunnan, China

Apple is the largest fruit tree crop in the world, and China is the largest apple-producing County in the world. Zhaotong, Yunnan Province is a typical cold and mountainous apple-producing area in China. However, apple production is threatened by diseases during the entire growing season, and among them, apple leaf spot and fruit black spot are severe. In previous reports, the main pathogen causing apple leaf spot and fruit black spot was Alternaria sp. (Lior, et al, 2017), while different pathogens were identified. In the current study, seven red Fuji apple fruit with typical black spot samples were collected randomly in Dongda company orchard, Sujiayuan town, Zhaotong, Yunnan on March 25, 2021. The spots on the surface of these apples appear rounded, the diseased parts turn brown or black in colour and the flesh became soften and rotten. The tissues of fruit epidermis at the edge between diseased and healthy parts were cut, soaked in 75% alcohol for 30 s, washed with sterile water three times, and air-dried. Five pieces of tissue were placed on PDA medium amended with rifampicin (50 mg/ml) and incubated in the dark at 25 ℃ for 3-5 days. After colonies grew, mycelial clumps were picked out from the edges of the colonies, transferred to new PDA plates, and incubated at 25 ℃ for 6 days. The diameter of the colonies reached up to 5.7 cm. A representative isolate was retained for further work and was named P6-3-1. The hyphae were white and dense at an early stage, the culture medium on the underside became yellow and the middle parts of the colonies were darker. With maturity, hyphae were clumped, became red with other colors interspersed, and the medium became dark red. Light brown spores were produced, with more vertical septa and fewer transverse septa. Two to three transverse septa were generally observed with obvious constriction at the transverse septa. Average spore size was 22.83 µm ± 2.04 µm × 14.58 µm ± 1.97 µm. DNA was extracted from mycelium, purified and amplified with two pairs of primers, ITS1/ITS4 (White et al. 1990) and gpdF/gpdR (Marcos P. S. Câmara, et al. 2002). The PCR products were sequenced and deposited in GenBank (accession NO.OK560128 and OK627661 ). The similarity of ITS sequences between the isolate and MH843733 (Stemphylium eturmiunum strain ST14) was 100%, and that of gpd sequences between the isolate and MH843728 (Stemphylium eturmiunum strain ST20) was 100%. The maximum parsimony method of Mega7.0 was used and demonstrated that the studied isolate converged to the same branch as Stemphylium eturmiunum. Koch's postulates was applied to identify the pathogenicity of this isolate. A disc of P6-3-1-culture on PDA (5 mm in diameter) was placed on apple leaves and fruit wounds. Sterile PDA was used as a control. All plants were kept in a growth chamber at 25-30 ℃. Four days after inoculation, the disease spot was observed on the inoculated sites and fruit, and with the extension of incubation time, the diseased spots continue to grow, and the leaf spots were not limited by the veins. The pathogen was re-isolated from the inoculated leaves and fruit, satisfying Koch’s postulates. This pathogen can also cause postharvest rot of sweet cherry (Alice Spadoni, et al, 2020), postharvest rot on tomato (Prencipe Simona, et al, 2021), etc. This is the first report that Stemphylium eturmiunum can cause apple leaf spot and fruit black spot in Yunnan province, China. The apple black spot caused by Stemphylium eturmiunum was accurately identified. By distinguishing between the two similar diseases mentioned above, resistance to the host and management practices can be accrued based on the characteristics of the pathogen, its epidemiological pattern and the choice of an effective chemical fungicide.

Monitoring benzimidazole resistance in Phyllosticta citricarpa using a molecular assay targeting mutations in codons 198 and 200 of the beta-tubulin gene

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is an economically important disease, which is effectively controlled by repeated fungicide applications to protect fruit from infection. Systemic fungicides such as benzimidazoles are widely used for controlling CBS in South Africa, but the molecular mechanisms of benzimidazole resistance in P. citricarpa had not been investigated. Analysis of the nucleotide sequence of the beta-tubulin gene in P. citricarpa revealed mutations inducing three amino acid replacements in benzimidazole-resistant isolates when compared to that of sensitive strains. Amino acid replacements in benzimidazole-resistant isolates included the change of glutamic acid to either alanine or lysine at codon 198 of the beta-tubulin gene and the change from phenylalanine to tyrosine at codon 200. All three mutations were previously implicated in benzimidazole resistance in several fungal pathogens. A polymerase chain reaction (PCR) assay was designed to amplify a portion of the beta-tubulin gene, which is subsequently sequenced to identify benzimidazole resistance in P. citricarpa. This PCR and sequence assay was found to be a more rapid and reliable method for detecting resistance compared to the fungicide-amended plate tests and is valuable for monitoring the occurrence of benzimidazole-resistant P. citricarpa and for assessment of the need for alternative CBS management practices.

Neopestalotiopsis rosae Causing Black Spot on Leaf and Fruit of Pecan (Carya illinoinensis) in China

Pecan (Carya illinoinensis K. Koch) is very productive and brings good economic returns for mountain dwellers. In recent years, symptoms were observed on both leaves and fruits of pecan in orchards, Fuyang, Anhui Province, and Jiande, Zhejiang Province. On the leaf, spots, rust-colored, long shuttle to irregular shaped, and 1–3 mm in size, firstly appeared between veins. The color of area around the spot gradually changed from green to yellow making a yellow halo. Then, some spots often merged in to a big lesion. On the fruit, symptoms firstly appear as irregular and black spots. The spots gradually spread to most part of the fruit and the core of the fruit turned black at the late stage of pathogenesis. Normally, the leaf infection rate was less than 10%, whereas infection rate of fruit could reach 5–15% which leads to drastic quality reduction and significant yield losses. To isolate the pathogens, fresh infected tissues were cut from samples, disinfected and cultured at 2% water agar at 28 oC till colony could be spotted. Fungal isolates were obtained by mycelia tip transferring. Similar cultural and morphological characters showed on 39 of the total 45 isolates. The mycelia grew rapidly in PDA plates, and colony diameter could reach 8.0 cm after culture at 25 oC in dark for 5 days. Mycelia on surface of the colony were cotton-like and showed obvious growth rings, and turn yellow from the middle on aged cultures. Pycnidia, brown to dark in color, began to form on PDA after 15 days’ culture in the dark. Conidia were fusoid, straight to slightly curved, 4-septate, 23.5–26.3×7.6–9.0μm, average 24.9×8.3μm (n=60) in size. The basal cell was hyaline, conic with a truncate base and thin-walled, average 4.4–5.1μm long (n=60). The three median cells were doliiform and brown with darker septa. The apical cell was hyaline, cylindrical, with 2–3 tubular apical appendages arising from the apical crest, unbranched, filiform, average 21.7–29.2 μm(n=60) long. The cultural and morphological characters of the strains matched the previous descriptions of Neopestalotiopsis sp. (Maharachchikumbura et al., 2014). Their 3 partial genes, ITS, β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1-α), were amplified and sequenced with primers (Lee et al., 2006; Maharachchikumbura et al., 2014). These sequences were submitted in GenBank with accession number of MW504791 (ITS), MW633956 (TEF1-α), and MW633958 (TUB2), seperately. The BLAST results against the GenBank database showed more than 99% identity with sequences claimed from Neopestalotiopsis sp. The multilocus phylogenetic analysis of the three genes showed the isolate was closest with N. rosae. The strains were finally identified as N. rosae by morphological and molecular analysis. Pathogenicity testing was performed by spreading 20μl conidia suspension drop (1 × 106 conidia ml-1) on surface of wounded or unwounded leaves and fruits. The pecan variety MaHan was used for inoculation. Wounds were made by scratching with sterile needles, and then a drop of 20μl conidia suspension was spread on the wounded area. For unwounded inoculation, a drop of 20μl conidia suspension was spread without wound procedure. The same amount of water drops were spread on the surface of wounded and unwounded tissues as controls. The plant parts were wrapped with PE films to keep moisture after drop spreading. Ten trees were selected for inoculation, and 10 leaves and fruits were randomly selected separately for wounded and unwounded inoculation and corresponding controls in each tree. The similar symptoms appeared from 3 dpi on leaves and 9 dpi on fruits, and finally showed on more than 90% wounded inoculated leaves or fruits and on less than 50% after unwounded inoculation, while controls remained symptomless. N. rosae was consistently re-isolated from inoculated parts with symptoms, while not from the control. Therefore, N. rosae was confirmed as the pathogen causing black spot on leaf and fruit of pecan (C. illinoinensis). To the best of our knowledge, this is the first report of N. rosae causing black spot disease of pecan (C. illinoinensis). In recent years, both the infection area and severity of this disease grow rapidly, which means the potential of the disease to become a big problem in local orchards.

Enemy or ally: a genomic approach to elucidate the lifestyle of Phyllosticta citrichinaensis

Members of the fungal genus Phyllosticta can colonize a variety of plant hosts, including several Citrus species such as Citrus sinensis (orange), Citrus limon (lemon), and Citrus maxima (pomelo). Some Phyllosticta species have the capacity to cause disease, such as Citrus Black Spot, while others have only been observed as endophytes. Thus far, genomic differences underlying lifestyle adaptations of Phyllosticta species have not yet been studied. Furthermore, the lifestyle of Phyllosticta citrichinaensis is ambiguous, as it has been described as a weak pathogen but Kochs postulates may not have been established and the presence of this species was never reported to cause any crop or economic losses. Here, we examined the genomic differences between pathogenic and endophytic Phyllosticta spp. colonizing Citrus and specifically aimed to elucidate the lifestyle of Phyllosticta citrichinaensis. We found several genomic differences between species of different lifestyles, including groups of genes that were only present in pathogens or endophytes. We also observed that species, based on their carbohydrate active enzymes, group independent of their phylogenetic association, and this clustering correlated with trophy prediction. Phyllosticta citrichinaensis shows an intermediate lifestyle, sharing genomic and phenotypic attributes of both pathogens and endophytes. We thus present the first genomic comparison of multiple citrus-colonizing pathogens and endophytes of the genus Phyllosticta, and therefore provide the basis for further comparative studies into the lifestyle adaptations within this genus.