Pink Pigment Research Articles

First Report of Fusarium oxysporum Causing leaf spot on Basella rubra in Malaysia.

Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is an edible perennial vine served as leafy greens in Malaysia. In May 2021, leaves with circular brown spots ranging from 3 to 10 mm wide with purple borders were found on B. rubra growing in Penampang (5°56'55.6"N 116°04'33.5"E), Sabah province. The disease severity was 80% with 10% disease incidence on 50 plants. As the disease developed, the lesions grew larger and they developed necrotic centers. Leaves with brown spot symptoms from five plants were collected from the field. Five leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022b), before incubation on water agar at 25°C. When five pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) at 25°C. After 5 days, fluffy white mycelia tinged with pink pigmentation showing on the underside of the colony were observed on PDA. Mycelia became violet in color as the culture aged. The isolates were incubated on carnation leaf agar at 25°C with a 12-hour light/dark photoperiod for 10 days. Sickle-shaped, thin-walled and delicate macroconidia (n= 30), predominantly 3 septate, ranging from 21.6 to 38.3 μm long by 2.7 to 4.2 μm wide in size were observed. Kidney-shaped, aseptate microconidia (n= 30) ranged from 6.2 to 11 μm long by 2.6 to 3.9 μm wide in size, and were formed on monophialides in false heads. Chlamydospores were detected both terminally and intercalarily, singly or in pairs, with smooth or rough walls. Genomic DNA was extracted from fresh mycelia of a representative isolate from Penampang based on Khoo et al. (2022a). The primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O'Donnell et al. 1998) were used to amplify the internal transcribed spacer (ITS) rDNA and translation elongation factor 1-α (TEF1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022b). The products were sent to Apical Scientific Sdn. Bhd. for sequencing. In BLASTn analysis, ITS sequence (OK469301) was 99% (506/507 bp) identical to isolate TSE07 (MT481761) of Fusarium oxysporum, and the TEF1α sequence (OM743433) was 100% (705/705 bp) identical to isolate BLBL5 of Fusarium oxysporum. The TEF1α sequence of Penampang was analyzed at the Fusarium MLST site (https://fusarium.mycobank.org/), and had 98% similarity to TEF1α of F. oxysporum (NRRL 22551). The pathogen was identified as F. oxysporum based on morphological (Leslie and Summerell 2006) and molecular data. A volume of 0.16 ml of spore suspensions (1 × 106 conidia/ml) were inoculated on a spot on each leaf of every three healthy B. rubra seedlings at the two-leaf stage. An additional three B. rubra seedlings were mock inoculated by pipetting sterile distilled water on similar aged leaf. The seedlings were maintained in a greenhouse at 25°C with a relative humidity of 80 to 90%. Six days after inoculation, all inoculated leaves exhibited the same symptoms as observed in the field, while the controls showed no symptoms. The experiment was repeated two more times. The reisolated fungi had the same morphology and DNA sequences as the original isolate obtained from the field samples, completing Koch's postulates. F. oxysporum has been reported previously in Bangladesh and India causing leaf spot disease on B. rubra (Dhar et al. 2015; Shova et al. 2020). To our knowledge, this is the first report of F. oxysporum causing leaf spot on B. rubra in Malaysia. The identification of leaf spot caused by F. oxysporum will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.

Photo-physiology of healthy-looking and diseased/health-compromised hard corals from Mauritius Island, Western Indian Ocean

Abstract. Jogee SY, Jeetun S, Ricot M, Taleb-Hossenkhan N, Mattan-Moorgawa S, Kaullysing D, Riemann P, Blanc L, Casareto BE, Suzuki Y, Bhagooli R. 2023. Photo-physiology of healthy-looking and diseased/health-compromised hard corals from Mauritius Island, Western Indian Ocean. Indo Pac J Ocean Life 7: 27-37. The spatial photo-physiological responses of in hospite zooxanthellae in hard corals, including coenosarc and polyps, healthy-looking and affected parts in four coral diseases, namely Brown Band, Black Band, Skeletal Eroding Band and White Band on the coral Acropora muricata, and two health-compromised conditions such as the Pink Pigmentation Response and its differentiated morphology, the Pink Line Syndrome, on the coral Porites were investigated using the Imaging-PAM fluorometry. A significantly lower Fv/Fm was observed in case of Black Band, White Band, Brown Band and Pink Pigmentation Response affected parts compared to the healthy-looking parts. The Fv/Fm had the highest decline in Brown Band disease. Both the polyps and coenosarc had significantly lower Fv/Fm in White Band and Brown Band diseased parts compared to their healthy-looking parts. The rETRmax did not change significantly between diseased/health-comprised parts and healthy-looking parts. NPQmax declined significantly in White Band, Black Band and Pink Pigmentation Response cases. ? and ? generally did not tend to be affected in diseased/health-compromised conditions. The photo-physiology of in hospite zooxanthellae was least affected in Pink Line Syndrome. These findings suggest that diseased/health-compromised parts of corals behave differently in terms of their photo-physiology in different diseased and health-compromised coral conditions in important reef-building corals species such as A. muricata and Porites species, with important implications for the productivity and thus adaptive management of coral reefs in a globally warming ocean.

Melanin synthesis gene Aapks contributes to appressorium formation, stress response, cell well integrity and virulence in Alternaria alternata

Melanin, as a common pigment, performs diverse biological function related to growth and development, environmental stress and pathogenicity in many pathogenic fungi. The polyketide synthase (Aapks) gene is a key gene in 1,8-dihydroxynaphthalene (DHN) melanin synthesis pathway, however, the regulatory roles of Aapks in pear fungal Alternaria alternata, on melanin synthesis, external stresses response, infection structure differentiation and pathogenicity are not clear. The pks gene was cloned and identified in A. alternata. Gene knockout experiment showed that ΔAapks mutant resulted in melanin deficiency and pink pigment production, reduced sporulation and abnormal spore and hyphae. Hypersensitivity to cell wall and oxidant stress factors were also found in ΔAapks, especially the impaired cell wall of ΔAapks was observed after CFW staining, and chitinase activity and related genes expression Aachs4 and Aachs6 were also decreased in ΔAapks. Interestingly, the ΔAapks mutant delayed infection structural differentiation induced by the different physicochemical signals. The virulence of ΔAapks strain was reduced after wound-inoculated pear fruit and non-wound-inoculated tobacco leaves. The above findings suggested the Aapks plays a vital regulatory role in response to stresses, infection structure formation, cell wall integrity and virulence in A. alternata by catalyzing the melanin synthesis.

Isolation and Characterization of Pigment Producing Bacteria from Solid Food Waste Samples

Due to increasing advertence of hazards of chemical colours, biocolours produced by pigment producing microorganisms can be helpful option to be used commercially. In the current study it was attempted to isolate pigment producing bacteria from solid food waste samples which are rich in microflora. Three isolates designated as IS-1, IS-2 and IS-3 were obtained which were later tentatively identified as Staphylococcus roseus (red pigment), Staphylococcus saprophyticus (pink pigment) and Micrococcus luteus (lemon yellow pigment), respectively on the basis of morphological and biochemical characteristics. Each pigment was further extracted and its antimicrobial activity was checked against Staphylococcus aureus, Candida albicans, Klebsiella pneumoniae and Pseudomonas aeruginosa. The pigment produced by Staphylococcus roseus showed antimicrobial activity against Staphylococcus aureus and Candida albicans. Hence, this red pigment has potential to be used in food industry and also in skin creams. Further work on this pigment for its feasibility in above use is in process.

Soil microplastics – current research trends and challenges: preliminary results of the earthworm Eisenia fetida impact on glitters

Abstract This paper shortly introduces the topic of soil microplastics by presenting a comprehensive review of the current state of the art in this field. It focuses mainly on the role of primary microplastics, particularly glitters, due to their large surface-to-volume ratio. From conclusions about current challenges and research directions, after analysis of the crucial “knowledge gaps “, one can point out the lack of a detailed description of the influence of microplastics on biota. The earthworms are promising model organisms that may play in soil ecosystems a similar role to the bivalves in waters. Thus, this mainly review paper was enlarged by the preliminary results of studies on glitters and bioglitters naturally aged in milli-Q water and soil with Eisenia fetida. Fourier-transform Infrared (FTIR, microscopy in the reflectance mode) and Raman spectroscopy (780 nm) were used to identify the subsequent notable changes in studied materials. The presence of the polymer (PET) core in the standard glitter particles is confirmed. In addition, the leakage of dyes from bioglitters was observed within the 9-week experiment. Tested bioglitters decompose in a slightly different way. Blue and pink pigments had entirely different stability as only one disappeared without a trace under UV light (blu), and the other remained stable in solution, confirmed by UV-Vis spectra. The nephelometry shows sedimentation of glitters after turbulent agents in the environment. This paper presents the natural weathering of primary microplastics in the presence of Eisenia fetida.

Genome analysis of Erwinia persicina reveals implications for soft rot pathogenicity in plants.

Soft rot disease causes devastating losses to crop plants all over the world, with up to 90% loss in tropical climates. To better understand this economically important disease, we isolated four soft rot-causing Erwinia persicina strains from rotted vegetables. Notably, E. persicina has only recently been identified as a soft rot pathogen and a comprehensive genomic analysis and comparison has yet to be conducted. Here, we provide the first genomic analysis of E. persicina, compared to Pectobacterium carotovorum, P. carotovorum, and associated Erwinia plant pathogens. We found that E. persicina shares common genomic features with other Erwinia species and P. carotovorum, while having its own unique characteristics as well. The E. persicina strains examined here lack Type II and Type III secretion systems, commonly used to secrete pectolytic enzymes and evade the host immune response, respectively. E. persicina contains fewer putative pectolytic enzymes than P. carotovorum and lacks the Out cluster of the Type II secretion system while harboring a siderophore that causes a unique pink pigmentation during soft rot infections. Interestingly, a putative phenolic acid decarboxylase is present in the E. persicina strains and some soft rot pathogens, but absent in other Erwinia species, thus potentially providing an important factor for soft rot. All four E. persicina isolates obtained here and many other E. persicina genomes contain plasmids larger than 100 kbp that encode proteins likely important for adaptation to plant hosts. This research provides new insights into the possible mechanisms of soft rot disease by E. persicina and potential targets for diagnostic tools and control measures.

Physicochemical Properties and Antibiosis Activity of the Pink Pigment of Erwinia persicina Cp2

The control and management of fungal diseases is a worldwide problem. A variety of microbial pigments have excellent antibacterial effects, and naturally occurring bacterial pigments may help in tackling fungal diseases. In order to explore the basic properties and biological functions of the pink pigment produced by Erwinia persicina Cp2, we used organic solvents to extract the pink pigment, analyzed the physicochemical properties of the pigment, determined the chemical composition using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and selected five pathogenic fungi to study the inhibitory effects of the pink pigment. The results showed that the main component of the pink pigment was usambarensine, which had a good light stability and a good temperature stability at room temperature (<40 °C), but the influence of the oxidant on its activity was greater than that of the reductant; simultaneously, we found that strong acids, strong alkalis, Cu2+, and Zn2+ all greatly affect the stability of the pink pigment, while Fe2+ and Fe3+ made the pigment darker. Meanwhile, the pigment could exert a good inhibitory effect against four plant pathogenic fungi: Alternaria solani, Sclerotinia sclerotiorum, Rhizoctonia solani, and Fusarium proliferatum. However, the inhibition of Fusarium oxysporum. f. sp cucumerinum decreased significantly in the later stages. This study had detected the purification process and antifungal activity on five fungi of the pink pigment of Erwinia persicina Cp2. It lays a theoretical and practical foundation for the production of related biological agents.

Effect of Extrinsic Pigmentation on Dimensional Stability, Hardness, Detail Reproduction, and Color of a Silicone.

The aim of the study is to evaluate the effect of extrinsic pigmentation on the dimensional stability, hardness, detail reproduction, and color of a silicone after thermocycling. Sixty samples of MDX4-4210 silicone (Dow Corning Corporation Medical Products) with intrinsic pink pigment (H-109-P, Factor II) and intrinsic opacifier (TiO) were fabricated. Two groups were created: Group 1-only intrinsic pigmentation (H-109P, Factor II + TiO) (Control); and Group 2-intrinsic (H-109P, Factor II + TiO) and extrinsic (Tan FE - 215, Factor II) pigmentation. The following tests were performed for each group: dimensional stability, Shore A hardness, detail reproduction, and color. Readings for the tests were taken before and after thermocycling (2,000 cycles). For dimensional stability and hardness, two-way analysis of variance (ANOVA) was used. One-way ANOVA was used for the color test. In case of significant statistical difference, the Tukey test was applied (p <0.05). All samples achieved the same detail reproduction score, therefore, no statistical evaluation was performed. For the dimensional stability test, comparing the initial time with the final time, there was a significant contraction in both groups after thermocycling. For the hardness test, comparing the time points, only group 1 showed a significant reduction in hardness after thermocycling. Groups 1 and 2 scored 2 for the detail reproduction test, before and after thermocycling. Comparing group 1 with group 2, there was no significant difference for color change. Based on the tests performed, extrinsic pigmentation did not show a negative effect on silicone, and therefore it can be indicated. The results of the dimensional, hardness, detail reproduction and color evaluations of the MDX4-4210 silicone were clinically acceptable in all cases in the groups with and without extrinsic pigmentation.

Occurrence of the Fusarium incarnatum-equiseti Species Complex Causing Fusarium Head Blight of Wheat in Mexico.

Fusarium head blight (FHB) is one of the most important diseases affecting wheat production worldwide. In Mexico, Fusarium boothii and F. avenaceum are the dominant species causing FHB of wheat (Cerón-Bustamante et al. 2018). During the 2017 to 2019 surveys, FHB symptoms were observed in wheat fields in the Highlands region of Mexico. Symptomatic spike samples were collected from 19 wheat fields in five states (Tlaxcala, Hidalgo, Puebla, Estado de México, and Morelos). Fusarium-like colonies were consistently isolated on potato dextrose agar (PDA) and 95 monoconidial isolates were obtained. Morphological features of seven isolates were consistent with the description of the Fusarium incarnatum-equiseti species complex (Xia et al. 2019). On PDA, colonies exhibited white and fluffy aerial mycelia, with diffused pink pigment on the reverse side after 7 days of incubation at 25℃. On carnation leaf agar (CLA), macroconidia (n = 100) were hyaline, falcate, with 3 to 6 septa, measuring 25.2 to 43.1 × 2.8 to 5.1 μm, and foot-shaped basal cell. Chlamydospores were ellipsoidal or subglobose and produced in chains. These seven isolates were selected for multilocus phylogenetic analysis and pathogenicity tests. Isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University under acc. nos. UACH428 to UACH434. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region, partial sequences of translation elongation factor 1-alpha (EF1-α) and the second largest subunit of RNA polymerase II (RPB2) genes were amplified, and sequenced with the primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. A phylogenetic tree, including published ITS, EF1-α, and RPB2 sequence data, was constructed for the Fusarium incarnatum-equiseti species complex (FIESC) based on Maximum Likelihood. Three species of the FIESC were identified into F. pernambucanum (five isolates), F. sulawesiense (one isolate), and F. clavum (one isolate). The sequences were deposited in GenBank with accession nos. OL347713 to OL347719 for ITS, OL365078 to OL365084 for EF1-α, and OL365072 to OL365077 for RPB2. The pathogenicity of the isolates was confirmed on wheat cv. Nana F2007 at the flowering stage in a glasshouse assay. The heads of 20 wheat plants were sprayed with a conidial suspension (1 × 105 spores/ml) of each isolate. Ten plants mock-inoculated with sterilized water served as the controls. All plants were placed in a moist chamber for 48 h. At 10 days after inoculation, typical FHB symptoms were visible on the inoculated plants, whereas the control plants remained asymptomatic. The pathogenicity test was repeated twice with similar results. The fungi were reisolated from the infected heads and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Previously, three isolates of Fusarium sp. belonging to the FIESC, were associated with FHB of wheat in Mexico (Cerón-Bustamante et al. 2018); however, this is the first report of F. pernambucanum, F. sulawesiense, and F. clavum causing FHB of wheat in Mexico and worldwide (Farr and Rossman 2021). Further studies should be focused on determining the distribution, prevalence, and toxigenic potential of the isolates of the FIESC associated with wheat diseases in Mexico.

Isolation, Characterization, and Optimization Studies of Bacterial Pigments

Recent concern for human safety and environmental protection has rekindled interest in natural pigment sources. In comparison to synthetic pigments, microbial pigments show better biodegradability and environmental compatibility and are used in a variety of applications ranging from food to cosmetics. The areas of attention for economical pigment synthesis include the identification of novel microbiological sources and improvement of process parameters. The purpose of this research was to screen and identify microbial isolates capable of generating pigments with antimicrobial activity from a variety of soil samples. A total of six pigment-producing bacterial sps were able to isolate from various soil samples such as bore well digging sites, river shores, river beds, forest areas, dumping yards using the enrichment culture technique. All the isolates were morphologically and biochemically identified as Micrococcus sp producing two-color pigments i.e., yellow and orange, Serratia sp producing red and pink color pigments, Salinococcus sp producing orange color pigment, and Exiguobacterium sp producing yellow color pigment respectively. During optimization studies maximum pigment production was observed at pH 7, agitation at 90 rpm (rotations per minute) and 120 rpm, the temperature of 30°C and 37°C, inoculum size up to 2% with NaCl concentration of 2%, 4%, and 6% respectively. Optimization of nutritional parameters such as carbon source and nitrogen source it was found that glucose (1%) and yeast extract (0.1%) work the best. Extraction of the pigment from the fermented broth was done by solvent-solvent extraction method. UV-Visible spectrophotometry and Silica gel Thin-layer chromatography was used to detect the presence of carotene and prodigiosin in the extracted bacterial pigment. The crude bacterial pigments were tested for antimicrobial activity against clinical pathogens including E. coli, Klebsiella sp, Bacillus sp, Staphylococcus sp, and pseudomonas sp respectively. Among all the isolates, pigments of Micrococcus sp and Salinococcus sp showed comparatively good results. Further purification of the pigment will lead to discovering a promising drug in the pharmaceutical industry.

First Report of Fusarium avenaceum Causing Leaf Spot on Angelica sinensis in China.

Angelica sinensis (Oliv) Diels (Umbelliferae) is a popular Chinese herb that is mainly distributed in Gansu Province, China, accounting for more than 90% of the national output and sales. A survey for diseases of A. sinensis in Gansu Province in August 2019 found foliar disease with an incidence of 60 to100%, and severities ranging from 5 to 15%. The disease mainly occurred in late July and August. The initial symptoms included many light brown, small lesions, round or irregular in shape, which gradually increased in size. White mycelia was visible in the lesions. Severely affected leaves became chlorotic, withered and died. In the Angelica planting area in Weiyuan County (33°26'N, 104°02'E) diseased leaves from 20 plants were collected by the five-point sampling method (Zheng et al. 2018), and small samples (4 × 4 mm2) wee cut from the border between diseased and healthy tissue, successively sterilized with 75% ethanol for 30 sec, washed three times with sterilized water and dried on sterilized filter paper, and placed on potato dextrose agar plates. After 5 days at 25°C, five morphologically similar colonies were obtained. Colonies were somewhat round with pink overall and formed abundant fluffy white mycelium in the center. Conidia were solitary, macrospores slender, straight to slightly falcate with 2 to 6 septa, and ranged from 20.0 to 77.6 µm × 2.5 to 3.6 µm (n=50). The microspores were elliptical and ranged from 3.0 to 8.0 µm × 2.5 to 3.0 µm (n=5). The strong pink pigment was observed on the reverse side of the PDA culture. The morphological characteristics were consistent with the description of Fusarium avenaceum (Parikh et al. 2018; Jahedi et al. 2019). To further identify the strains, the internal transcribed spacer (ITS), β-tubulin, translation elongation factor 1α (EF1-α), and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4, Bt2a/Bt2b, EF1/EF2, and 5f2/7cr (Glass and Donaldson 1995; O'Donnell et al. 2010; White et al. 1990), respectively. The sequences of the five strains were identical, and that of representative strain K0721 were deposited in GenBank (ITS, MZ389899; TUB2, MZ398139; EF1-α, MZ388462; RPB2, MZ394004). BLAST analysis revealed that the ITS, β-tubulin, EF1-α, and RPB2 sequences were 100% (563/563), 100% (423/423), 99% (643/649), and 99% (930/935) homology, with those of F. avenaceum (KP295511.1, KY475586.1, KU999088.1, and MH582082.1), respectively. A multigene phylogenetic tree was inferred by Maximum likelihood phylogenetic analyses based on the combined data set with ITS, EF1-α and RPB2. The strain K0721 was clustered with F. avenaceum. Pathogenicity tests were performed on five 1-month-old healthy plants in plastic pots (20 cm. diam.) with sterilized soil. Each was sprayed with 50 μl of a conidial suspension (1×104 conidia/mL), and 5 healthy plants were sprayed with sterile water as controls. Small lesions were observed after 5 days at 25℃ in a greenhouse. Symptoms were similar to those observed under field conditions. Control plants remained symptomless. Six isolates were reisolated from infected leaves and all confirmed to be F. avenaceum based on morphological observations and molecular identification. To our knowledge, only Septoria anthrisci has been previously reported as a pathogen of A. sinensis leaf spot (Wang et al. 2018), and this is the first report of F. avenaceum causing this disease. This discovery needs to be considered in developing and implementing disease management programs in A. sinensis production.

First Report of Erwinia persicina Causing Stalk Rot of Celery in China.

Species belonging to the genus Erwinia cause diseases in many economically important plants. In May 2021, celery (Apium graveolens var. dulce) plants (cv. Queen of France) showing soft rot symptoms were observed in greenhouses in Boye County, Baoding, Hebei Province (North China). Disease symptoms began with pinkish water-soaked lesions on the midrib of celery stalks, but at the same time the leaves and root were asymptomatic; and the infected celery plants rapidly developed brownish rotten stalks. The disease incidence in two greenhouses (0.15 ha in size) was more than 50%. Affected celery stalk pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were immersed in 200 µl 0.9% saline for 15 min. Aliquots (20 μl) of two tenfold dilutions of the tissue specimen soaking solution were plated onto Luria-Bertani (LB) medium and incubated at 28°C for 24 h. Single colonies were picked and restreaked onto LB agar three times for purity. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R and sequenced. Result of blastn analysis of the 16S rDNA amplicons (MZ614654, MZ614655) indicated that the bacterial isolates (BY21211 and BY21212) belonged to the genus Erwinia. Housekeeping genes including mdh, gapA, icdA, rpoS, acnA and proA were also amplified using a set of PCR primers (Ma et al. 2007) followed by sequencing (MZ643221-MZ643232). No sequence variation was observed at any MLSA locus between isolates BY21211 and BY21212. To determine the species of the Erwinia isolate BY21211 and BY21212, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). The result of phylogenetic analysis showed that the celery stalk rot isolate BY21211 and BY21212 was clustered with E. persicina type strain NBRC102418T (Hao et al. 1990). When celery plants (cv. Queen of France) have eight to nine true leaves, plants were inoculated with the isolate BY21211 by injecting 20 µl of bacterial suspensions (107 CFU·mL-1) into the celery stalks, and negative controls were injected with 20 µl of 0.9% saline. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacterial-inoculated seedlings showed disease symptoms resembled to those observed in greenhouses. Bacterial colonies were obtained from the infected stalks and were identified using the same PCR primers of housekeeping genes as described above. Therefore, E. persicina isolate BY21211 fulfill Koch's postulates for stalk rot of celery. Isolates BY21211 and BY21212 produce water-soluble pink pigment on sucrose-peptone agar. These isolates were gram negative and rod shaped, negative for oxidase, urease, indole production, gelatin liquefaction and acid production from xylose and glycerol. They were positive for catalase, citrate utilization, acid production from sorbitol, raffinose, glucose, arabinose, cellobiose, rhamnose, maltose, saccharose, inositol, lactose and esculin (hydrolysis). E. persicina has been reported to cause pink seed, crown and stem rot, soft rot or leaf spot on many plant hosts including pea (Pisum sativum), soybean (Glycine max), common bean (Phaseolus vulgaris), lucerne (Medicago sativa), barley (Hordeum vulgare), onion (Allium cepa), garlic bulbs (Allium sativum), tomato (Lycopersicon esculentum) and cucumber (Cucumis sativus) (Hao et al. 1990; González et al. 2005, 2007; Zhang and Nan 2014; Gálvez et al. 2015; Cho et al. 2019; Kawaguchi et al. 2021). To our knowledge, this is the first report of E. persicina causing stalk rot in celery. Stalk rot of celery has increased in prevalence over recent years in the Baoding region, it can cause significant yield loss and no cultivar has been found to be resistant to this disease so far. The stalk rot poses significant threat to local celery production, and further research on epidemiology and disease management options is needed.

Fusarium incarnatum-equiseti Species Complex Causing Root Rot Disease on Leymus chinensis in China.

Leymus chinensis (Trin.) Tzvel. is a rhizomatous grass widely grown in the grasslands of Eurasia. With strong fertility and stress resistance, L. chinensis makes an excellent pasture and mowing grass, contributing to animal husbandry and thus playing an important role in the local economy of the northern grassland area in China (Baoyin et al. 2014). During August to September 2019, diseased roots of L. chinensis were collected from an artificially planted grassland (40°47'44" N, 111°43'58″ E, alt. 1049 m) in Shaerqin County, Hohhot, China. Infected plants were scattered across the field with disease incidence up to 2%. Symptoms observed were wilted plants and rotten roots. In order to identify the causal pathogen of root rot on L. chinensis, symptomatic pieces (5 × 5 mm) of grass roots were excised and surface sterilized with 75% ethanol for 3-5 s followed by 1% NaClO for 2-3 min, rinsed three times with sterile distilled water, and placed on water agar and incubated at 25°C for 3 days. The mycelia were cut and transferred onto potato dextrose agar (PDA) for subculture. A fungus was consistently isolated, and a strain, named LCH054, was obtained by hyphal tip culture. Culture developed as white and fluffy aerial mycelia, with diffused pink pigment on the reverse side of PDA after culturing at 25℃ for 7 days. A culture of LCH054 was transferred to carnation leaf agar (CLA) (Li et al. 2014) and incubated at 25°C for 10 days. Microconidia were absent but macroconidia were produced. Macroconidia were hyaline, sickle-shaped, and had 4 to 7 septa, 19.8 to 63.6 (mean 43.8) × 1.8 to 5.7 (mean 3.2) μm (n = 100). Chlamydospores were ellipsoidal or subglobose, with thick walls in clumps or chains. All morphological characteristics of LCH054 resembled Fusarium equiseti (Leslie and Summerell 2006). The primers of the internal transcribed spacer (ITS) region (White et al. 1990) and translation elongation factor 1α gene (TEF-1α) (O'Donnell et al. 1998) were used to amplify the isolate, and the fragments were sequenced. BLASTn search in the NCBI database using the ITS and TEF-1α sequences revealed 99 to 100% similarities with F. equiseti. BLAST analysis of the ITS and TEF-1α sequencies in the FUSARIUM-ID database showed them to have 99.21% (500 bp out of 504 bp) and 99.52% (622 bp out of 625 bp) similarities with the Fusarium incarnatum-equiseti species complex (FIESC) (strain NRRL 45997) (O'Donnell et al. 2009), respectively. The ITS and TEF1-α sequences were deposited in GenBank as accession numbers MT937067 and MT947530, respectively. The strain LCH054 was identified as a member of the FIESC based on morphological and molecular characteristics. For the pathogenicity test, one hundred of L. chinensis seeds were planted into five pots (12 cm [diameter]) × 15 cm [high]) and kept in a greenhouse under a 16-h photoperiod with temperatures of 20-25°C and 40% relative humidity. The conidial suspension of LCH054 was prepared by washing 7-day old fungal culture grown on CLA medium using sterile deionized water. Conidia were filtered through three layers of sterile cheese cloth, counted, and adjusted to 1 × 105 conidia/ml with a hemocytometer. Forty 1-month-old healthy plants (four pots) were inoculated with 400 ml of conidia suspension using the root drenching method, whereas the inoculum was replaced with 100 ml sterile water on control plants (one pot). Fourteen days after inoculation, all inoculated plants showed the typical symptoms of root rot identical to those observed in the field, whereas the control plants remained healthy. LCH054 was re-isolated from the inoculated plants and identified by the morphological and molecular approaches as described above. To the best of our knowledge, this is the first report of root rot caused by F. incarnatum-equiseti on L. chinensis in China as well as worldwide. The presence of the pathogen could cause significant economic losses in L. chinensis production. For this reason, strategies for the management and control of this disease should be developed and implemented.

Tyrosine promotes anthocyanin biosynthesis in pansy (&lt;i&gt;Viola × wittrockiana&lt;/i&gt;) by inducing ABA synthesis

<italic>Viola × wittrockiana</italic> (pansy) is an important ornamental plant, particularly during winter and spring. In previous studies, we found that the tyrosine decarboxylase gene of pansy (<italic>VwTYDC</italic>) was expressed differently in blotched and non-blotched areas of pansy petals, suggesting that tyrosine may have a role in anthocyanin biosynthesis. In this study, we found that virus-induced gene silencing of <italic>VwTYDC</italic> caused an accumulation of pink pigmentation in pansy petals. Likewise, exogenous tyrosine treatment (TYRT) induced the formation of black stripes in non-blotched petal areas. Metabolome analysis indicated that the contents of two anthocyanins, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, increased significantly in the TYRT areas. RT-qPCR results revealed that the anthocyanin-related genes <italic>VwHCT</italic>, <italic>VwC3′H</italic>, <italic>VwCHS</italic>, and <italic>VwUGT</italic> were upregulated in the same areas. Transcriptome analysis revealed that four genes involved in the abscisic acid (ABA) biosynthesis pathway (<italic>VwNCED</italic>, <italic>VwABA2</italic>, <italic>VwAAO3</italic>, and <italic>VwCYP707A</italic>) were significantly upregulated in the same TYRT areas. ABA contents were measured by ESI-HPLC-MS/MS, and ABA content was significantly higher in TYRT areas than in control areas. In addition, when exogenous ABA was spread onto non-blotched petal areas, anthocyanin biosynthesis genes were upregulated just as with tyrosine. Thus, transcriptome and metabolite analyses revealed a possible novel regulatory network for anthocyanin biosynthesis in which tyrosine induces ABA synthesis and ABA then promotes anthocyanin biosynthesis in pansy petals.

First Report of Fusarium equiseti, Causing Fruit Rot Disease of Watermelon in Malaysia.

Watermelon (Citrullus lanatus) accounts for almost 13% of all tropical fresh fruit production in Malaysia. They are grown, mostly in Johor, Kedah, Kelantan, Pahang, and Terengganu areas of Malaysia on 10,406 ha and yielding 172,722 Mt. In 2019, a new fruit rot disease was observed in two major production areas in Peninsular Malaysia. Disease symptoms included water-soaked brown lesions on the fruit surface in contact with the soil. The lesions enlarged gradually and ultimately covered the whole fruit with white mycelium leading to internal fruit decay. Disease surveys were conducted in December 2019 and November 2020 in fields at Kuantan, Pahang and Serdang, Selangor. Disease incidence was 10% in 2019 and 15% in 2020. Infected fruits were collected and washed under running tap water to wash off adhering soil and debris. Fruit tissue sections 1 to 2 cm in length were surface sanitized with 0.6% sodium hypochlorite (NaOCl) for 3 min. and washed twice with sterile distilled water. The disinfected air-dried tissues were then transferred onto potato dextrose agar (PDA) media and incubated at 25±2℃ for 3 days. Fungal colonies with whitish mycelium and pink pigment isolated using single spore culture. The pure cultures were placed onto carnation leaf agar (CLA), and the culture plates were incubated at 25±2℃ for 15 days for morphological characterization. On CLA, macroconidia were produced from monophialides on branched conidiophores in orange sporodochia. Macroconindia were thick-walled, strong dorsiventral curvature, 5 to 7 septate with a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 21.9 to 50.98 μm long and 2.3 to 3.60 μm wide. Typical verrucose thick chlamydospores with rough walls were profuse in chains or clumps, sub-globose or ellipsoidal. Based on morphological characteristics they were identified as Fusarium equiseti (Leslie and Summerell 2006). Molecular identification of both U4-1 and N9-1 pure culture isolates were carried out using two primer pair sets; internal transcribed spacer (ITS) ITS-1/ ITS-4 and translation elongation factor 1 alpha (TEF1-α) (EF-1/EF-2). A Blastn analysis of the ITS gene sequence of U4-1(MW362286) and N9-1 (MW362287) showed >99% similarity index to the reference gene sequence of F. equiseti isolate 19MSr-B3-4 (LC514690). The TEF1-α sequences of U4-1 (accession no. MW839563) and N9-1 (accession no. MW839564) showed 100% identity; with an e-value of zero, to the reference gene sequence of F. equiseti isolate URM: 7561 (accession no. LS398490). Each isolate also had a >99% identity with isolate NRRL 34070 (accession no. GQ505642) in Fusarium MLST database that belongs to the F. incarnatum-equiseti species complex (O'Donnell et al. 2015). Based on phylogenetic analysis of the aligned sequences (TEF1-α) by the maximum likelihood method, the U4-1 and N9-1 isolates were confirmed to be F. equiseti as was reported in Georgia, USA (Li and Ji 2015) and in Harbin, Heilongjiang Province, China (Li et al. 2018). Finally, the two pure culture isolates of U4-1 and N9-1 were used to fulfill Koch's postulates. Stab inoculations of five healthy watermelon fruits (cv. 345-F1 hybrid seedless round watermelon) were performed with a microconidial suspension of individual isolates (4x106 spores/mL). Five control fruits were stabbed with double distilled water. The inoculated fruits were incubated under 95% relative humidity at a temperature of 25±2℃ for 48 h followed by additional incubation inside an incubator at 25±2℃ for 8 days. Ten days post-inoculation, the control fruits showed no disease symptoms. However, inoculated fruits exhibited typical symptoms of fruit rot disease like water-soaked brown lesions, white mycelium on the fruit surface and internal fruit decay, which is similar to the farmer's field infected fruits. The suspected pathogen was successfully re-isolated from the symptomatic portion of inoculated fruit and morphologically identified for verification. To our knowledge, this is the first report of F. equiseti causing fruit rot of watermelon in Malaysia. Malaysia exports watermelon year-round to many countries around the world. The outbreak of this new fruit rot disease could potentially pose a concern to watermelon cultivation in Malaysia.

First Report of Fusarium Root and Stem Rot Caused by Fusarium oxysporum f. sp. radicis-cucumerinum on Greenhouse Cucumbers in Saudi Arabia.

Cucumber (Cucumis sativus L.) is an important vegetable crop in Saudi Arabia. During May 2018, 45 - 60% of 5-month-old cucumber plants showed symptoms of a previously unknown wilt in commercial greenhouses around Al Kharj area of Riyadh region. Symptoms consisted of crown and root rot, wilting and stem disintegration, along with yellowish brown to brown external discoloration extended throughout the affected tissues. As the disease progressed, a pinkish-orange mycelial growth was often observed at the basis of affected stems while vessels were discolored. Subsequently, the affected plants were collapsed and died. Crown, stem, and root fragments (4 × 4 mm) were cut from symptomatic tissues, surface sterilized in 2.5% NaOCl, cultured on potato dextrose agar (PDA) with 25 mg/liter of streptomycin sulfate, and incubated at 26°C in darkness for 6 days. Single-spored cultures produced white mycelium with pink, white, or purple pigmentation in the center. The mycelium produced sporodochia. Macroconidia were mainly slightly curved with three to five septa. Microconidia were single-celled oval and produced on short lateral phialides. Chlamydospores were single or in short chains. Morphologically, the isolated fungus was characterized as Fusarium oxysporum (Leslie and Summerell 2006). To further confirm the fungus identification, DNA was extracted from a single-spored culture. Three different fungal nuclear regions of internal transcribed spacer (ITS), elongation factor 1-α, (TEF1-α) and the second largest subunit of DNA-directed RNA polymerase II (rpb2) with the following primers: ITS4 and ITS5 (White et al. 2017), EF-1 and EF-2 (O'Donnell et al. 2008), and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. The ITS, TEF1-α, and rpb2 sequences of the isolate FCKSU17 were submitted to GenBank (MT232918, MW471131, and MW449833 respectively). Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and rpb2 sequences using MEGA7 placed this strain in the F. oxysporum clade. To confirm the forma specialis radicis-cucumerinum, amplification with the specific primers ForcF1/ForcR2 was conducted (Lievens et al. 2007). The amplified fragment (∼ 250-bp) was sent for sequencing, and the sequence was submitted to GenBank (MW471132). BLASTn analysis of the sequences showed 100% identity with F. oxysporum radicis-cucumerinum (KP746408). To fulfill Koch's postulates, pathogenicity test was conducted on 7-day-old plants of cucumber cultivar Beit Alpha grown into pots filled with soil mix (2:1 sandy loam-peat moss, vol/vol). The plants were inoculated through drenching with 100 ml of conidial suspension in sterile distilled water (106 spores/ml) per pot. Control plants were treated with sterile distilled water. Each treatment included 10 replicates (pots), with two plants per pot. The pathogenicity test was repeated once. Cucumber plants inoculated with the fungus showed early wilting symptoms within the first 2 weeks post inoculation. At the 6th week post inoculation, 90 to 100% of the inoculated plants developed typical symptoms. No symptoms were observed on the control plants. The pathogen was successfully re-isolated from the inoculated wilted plants and identified morphologically. To our knowledge, this is the first report of F. oxysporum f.sp. radicis-cucumerinum on cucumber in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses on cucumbers in Saudi Arabia.

The effect of mineralizer addition and temperature on synthesis of malayaitepink pigment

PurposeThis study aims to the effect of NaF, Li2CO3 and H3BO3 minerals was investigated, and the best mineralizer was found to be H3BO3. Furthermore, the effect of temperature was investigated, and the synthesized samples were calcined at temperatures of 1100, 1200 and 1300 °C to select the optimum calcination temperature.Design/methodology/approachThis study was aimed at thoroughly investigating the effects of mineralizer addition and temperature on the synthesis of malayaite pink pigment based on raw materials of SnCl2-SiO2-Ca(OH)2-K2Cr2O7. To this end, the optimization of the synthesis parameters such as mineralizer addition and temperature was completely perused.FindingsThe optimum temperature was 1300 °C, and the color efficiency of pigments was evaluated by colorimetric (CIE L*a*b* system) analysis, and these parameters were close to those of industrial pigments.Originality/valueTo the best of the authors’ knowledge, for the first time, the effect of mineralizer addition and temperature on the synthesis of malayaite pink pigment was investigated through the sol-gel method. Herein, different parameters were optimized to propose a novel pigment with a much better performance.

First Report of Crown Rot Caused by Fusarium redolens on Wheat in Kazakhstan.

Fusarium crown rot, caused by several species within the genus, is a major constraint that results in significant losses in wheat production worldwide. In June 2019, diseased wheat plants with typical symptoms of crown rot, including discoloration on the first two or three internodes of the stem just above the soil line and stunted, dry rotted, and discolored roots were collected in several bread wheat fields during the maturity stage in Almaty, East Kazakhstan, and Karaganda Regions of Kazakhstan. For each field, approximately twenty tillers were randomly sampled. Symptomatic tissues were surface sterilized in 1% NaClO for 2 min, rinsed with sterile distilled water three times, air-dried in a laminar flow hood, and then transferred to Petri dishes containing one-fifth strength potato dextrose agar (PDA). After incubating in the dark at 23°C for 5 days, 79 single-spore isolates showing cultural and microscopic characteristics of Fusarium were obtained on PDA and Spezieller-Nährstoffarmer agar (SNA). Colonies were initially white but later produced a beige to pink diffusible pigment in PDA. Microconidia that formed on aerial monophialides were hyaline, 0 to 1 septum, oval- to kidney-shaped, and measured 4.3 to 10.3 × 1.9 to 3.4 µm (average 7.8 × 2.6 µm), whilst macroconidia were straight to slightly curved, 3 to 5 septate, and measured 18.7 to 38.8 × 2.9 to 6.6 µm (average 29.9 × 4.7 µm), with foot-shaped basal cells on SNA. Chlamydospores were present on PDA. Sequence analysis based on portions of translation elongation factor 1α (TEF1) and the nuclear ribosomal internal transcribed spacer region (ITS rDNA) loci with primers EF1/EF2 (O'Donnell et al. 1998) and ITS1/ITS4 (White et al. 1990) identified 29 of the 79 isolates as Fusarium redolens Wollenw. The sequences of the five representative isolates with 99.85% of similarity to those of F. redolens strains available in GenBank e.g., ITS (MT435063) and TEF1 (GU250584). The TEF1 (accession nos. MW403914-MW403918) and ITS rDNA (accession nos. MW397138-MW397142) sequences of the isolates were deposited in GenBank. The morphological features are consistent with the described features of F. redolens (Leslie and Summerell 2006). To confirm pathogenicity of the five isolates, five pre-germinated seeds of wheat cultivar Seri 82 were placed in a 9-cm-diameter pot filled with a sterile potting mix containing equal volumes of peat, vermiculite, and soil. An approximately 1-cm-diameter 7-day-old mycelial plug of each isolate was individually placed in contact with the seeds. Seeds were covered with the same potting mix, and then the pots were maintained for four weeks in a growth chamber at 23°C with a 12-h photoperiod. The experiment was conducted twice with three replicate 15-cm pots with 5 plants per pot. Controls were inoculated with sterile agar plugs using the same procedure. After four weeks, all the inoculated plants showed stunted growth with brown discoloration in most parts of the crown and roots, whereas no symptoms were observed in the control plants. The mean severity of the disease for each isolate was between 2.1 and 2.7 according to the scale of 1 to 5 described by Gebremariam et al. (2015). The pathogen was reisolated from crowns of diseased plants, but not from asymptomatic control tissues, and identified morphologically based on the methods described above, fulfilling Koch's postulates. Although several morphological features are shared by F. oxysporum and F. redolens, Baayen et al. (2001) showed that these species could be easily distinguished using molecular data. The pathogen was previously reported as F. redolens associated with crown rot of wheat in Turkey (Gebremariam et al. 2015) and Saskatchewan, Canada (Taheri et al. 2011). The presence of F. redolens causing crown rot is confirmed in the six wheat fields surveyed in Kazakhstan, for the first time. This pathogen may pose a risk for wheat production, and further studies needed to determine the impact on the crop in Kazakhstan.

Pedobacter endophyticus sp. nov., an endophytic bacterium isolated from Carex pumila.

An aerobic, Gram-stain-negative, weak-motile, short-rod-shaped bacterial strain, designated JBR3-12T, was isolated from halophyte Carex pumila plants, and its taxonomic position was investigated by using a polyphasic taxonomic approach. The strain produced a pink pigment on tryptic soy agar and grew optimally at 25 °C, pH 8 and in the presence of 3 % (w/v) NaCl. Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain JBR3-12T formed a lineage within the genus Pedobacter and was most closely related to Pedobacter sandarakinus DS-27T (98.0 %) and Pedobacter agri PB92T (97.6 %). The DNA G+C content of the genome was 41.3 mol%; the whole genome length was 5 426 070 bp. The major fatty acids of JBR3-12T were iso-C15 : 0, summed feature 3 (comprising C16 : 1 ω6c and/or C16 : 1 ω7c) and iso-C17 : 0 3-OH. The predominant polar lipid was phosphatidylethanolamine. The predominant quinone was menaquinone-7. Based on its phenotypic, phylogenetic and genotypic features, strain JBR3-12T is proposed to represent a novel species of the genus Pedobacter, for which the name is Pedobacter endophyticus sp. nov. The type strain is JBR3-12T (=KCTC 82363T=NBRC 114901T).

Muscle pigmentation in rainbow trout (Oncorhynchus mykiss) fed diets rich in natural carotenoids from microalgae and crustaceans

The amount of ingredients of marine origin used in salmonid feed industry is rapidly decreasing and fish meal and fish oil have been largely replaced by vegetable ingredients. New potential protein rich ingredients for aquafeed formulations should therefore be alternative to vegetable ones. The pattern of fillet pigmentation was thus assessed in rainbow trout (Oncorhynchus mykiss) fed for 15 weeks isoproteic (42%) and isolipidic (24%) diets deprived of fish meal where 10% of protein from a blend of vegetable ingredients was replaced by cyanobacteria and microalgae dried biomass (Arthrospira platensis, Tisochrysis lutea and Tetraselmis suecica) or Louisiana red swamp crayfish (Procambarus clarkii) meal. An automatic, repeatable, and objective image analysis tool for the integrated determination and direct visualization of fillet colour was applied to digital images. Colour of fillets was also measured with a chromameter, recording data as L*, a*, b* indexes and used to validate image analysis results. Fillet carotenoid and lipid contents were determined, and their pattern of variation was compared to that obtained by image analysis. Fillet colouring capacity of crayfish meal was also compared to that of commercial synthetic astaxanthin included in rainbow trout feed. To this last aim, test diets were administered for 12 additional weeks.The image analysis and colorimetric data consistently indicated that, even if characterized by a significantly higher carotenoid content respect to fish fed a vegetable diet, fillets of trout fed the cyanobacteria and microalgae including diets displayed an undesirable yellowish colour. P. clarkii meal instead resulted a promising functional ingredient to supplement plant protein-based diets for trout, in view of its lipid composition and astaxanthin content. In fact, despite a relatively low dietary carotenoid level due to low percentage of inclusion, a desirable pink pigmentation was clearly detectable in fillets of trout fed the crayfish meal-including diet, which resulted in colour differences which were less obvious relative to those of fish given the astaxanthin-supplemented feeds at the end of the additional 12 weeks.