Orange Pigment Research Articles

Documentation, investigation and analysis of a rare archaeological cartonnage object from the Egyptian Museum in Cairo, Egypt, using non-invasive methods

Technical and analytical investigations were carried out on an overhead mask of woman cartonnage from the Egyptian Museum in Cairo (catalogue 33279), which dated back to the Greco-Roman Period. Various non-invasive techniques were used in this study such as: AutoCAD program, multispectral imaging with Ultraviolet and Infrared Rays, optical microscope, X-ray fluorescence spectrometry (XRF), and Fourier transform infrared spectroscopy (FTIR). The structure of the cartonnage was unique and consisted of three distinctive layers, from the bottom to the top; a double layer from linen, a calcite-based plaster layer, and finally a polychrome paint layer. The study of the paint layer revealed the presence of blue, red, white, yellow, orange, brown and black pigments. Yellow color was identified as orpiment (As2S3) and yellow ochre (goethite α-FeO.OH + clay minerals) and blue as Egyptian blue (CaCuSi4O10). Two red shades were also detected of which the lighter is red lead (Pb3O4) and the darker is a mixture hematite (α-Fe2O3), red lead (Pb₃O₄) and calcite (CaCO3). Orange was identified as a mixture of orpiment (As2S3) and red lead (Pb3O4), white as white lead (PbCO3)2•Pb(OH)2,and black as magnetite (Fe3O4). The brown pigment, made up of red hematite, red lead, and black manganese, was detected for the first time in the pigment palette of ancient Egyptian cartonnage. The binding medium in linen layer was identified as Arabic gum. The study showed that cartonnage dated back to the Graeco-Roman Period because of the appearance of red lead, orpiment, and Egyptian blue. Moreover, the presence of lead in the components of Egyptian blue is considered evidence that ovens contain lead resulting in changing the manufacturing techniques of Egyptian blue.

Carotenoids: Role in Neurodegenerative Diseases Remediation.

Numerous factors can contribute to the development of neurodegenerative disorders (NDs), such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and multiple sclerosis. Oxidative stress (OS), a fairly common ND symptom, can be caused by more reactive oxygen species being made. In addition, the pathological state of NDs, which includes a high number of protein aggregates, could make chronic inflammation worse by activating microglia. Carotenoids, often known as "CTs", are pigments that exist naturally and play a vital role in the prevention of several brain illnesses. CTs are organic pigments with major significance in ND prevention. More than 600 CTs have been discovered in nature, and they may be found in a wide variety of creatures. Different forms of CTs are responsible for the red, yellow, and orange pigments seen in many animals and plants. Because of their unique structure, CTs exhibit a wide range of bioactive effects, such as anti-inflammatory and antioxidant effects. The preventive effects of CTs have led researchers to find a strong correlation between CT levels in the body and the avoidance and treatment of several ailments, including NDs. To further understand the connection between OS, neuroinflammation, and NDs, a literature review has been compiled. In addition, we have focused on the anti-inflammatory and antioxidant properties of CTs for the treatment and management of NDs.

Improved natural food colorant production in the filamentous fungus Monascus ruber using CRISPR-based engineering

Monascus pigments have various food industry applications and are pharmacologically active. Genome sequencing-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been implemented to increase pigment production in Monascus. To increase pigment production in M. ruber KACC46666, the CRISPR/Cas9 system was used to introduce mutations in two negative regulator genes (MpigI and MpigI′), among other genes involved in the Monascus pigment biosynthesis pathway. Dual single-guide RNAs were constructed to inactivate MpigI and MpigI′. After CRISPR/Cas9 inactivation, yellow, orange, and red pigment expression in the resulting △MpigI16-7 strain (among several Cas9-mediated mutants studied) was 2.5-, 12.4-, and 18.5-fold, respectively, higher than that in the wild-type strain. This study provides valuable information regarding CRISPR-guided metabolic engineering for natural colorant production.

First report of Fusarium oxysporum and Fusarium solani causing root rot on trifoliate orange rootstock in China.

Trifoliate orange (Poncirus trifoliata L) is a thorny tree of the Rue family, which is extensively used as citrus rootstock in China. In January 2021, several leaf yellowing, declining, and wilting citrus seedlings grafted on trifoliate orange rootstock with rotted main roots were observed in orchards located in Wuhan city, Hubei, China. In old orchards, the incidence of diseased roots was approximately 90%. Diseased roots from seven plants were collected and cut into small pieces (0.2 to 0.5 cm). These pieces were then surface-sterilized using 0.1% mercury bichloride for 3 min, 75% ethanol for 3 min, rinsed with sterile distilled water for several times, and then placed on potato dextrose agar (PDA) supplemented with 0.05% lactic acid (v/v), and incubated at at 25±2°C in dark. Fifty-threesingle-conidium isolates with morphological characteristics similar to Fusarium spp. were obtained (Leslie and Summerell 2006), which displayed two kinds of colony morphology. Thirty isolates showed white to orange-white abundant aerial mycelium in rings and acquired a yellow to orange pigmentation, tweenty-three isolates showed white to pink, fluffy aerial mycelium in rings and acquired an orange to red pigmentation. Isolate WG-1 and HrmY-9 from each group were used for future identification. The average colony growth rate of WG-1 and HrmY-9 on PDA was 0.95±0.06 and 0.69±0.11 mm/day, n=4, respectively. WG-1 produced numerous oval, unicellular microconidia without septa, 4.03-9.87×1.01-5.13 µm, n=80 and very few macroconidia with two to four septa, narrowed at both ends, 11.08-22.64×1.67-4.91 µm, n=30. HrmY-9 produced numerous curved macroconidia with three to four septa, 18.03-37.33×2.16-7.8 µm, n=80, microconidia were unicellular, oval, and 5.33-16.19×1.74-6.51 µm, n=50. Sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF-1α), and DNA-directed RNA polymerase largest subunit (RPB1) genes were amplified with the primers ITS1/ITS4, EF1a-F/EF1a-R, and RPB1-F5/RPB1-R8, respectively (White et al. 1990, O'Donnell et al. 1998, O'Donnell et al. 2010), sequenced and deposited in GenBank. Sequences of isolate WG-1 (GenBank accession No. ON045437, ON063232 and ON089664) and HrmY-9 (GenBank accession No. ON045438, ON063233 and ON089665) were 100% identical with the corresponding sequences of Fusarium oxysporum (OM876904, JF430180, and MT568959) and F. solani (MT605584, MK617767, and MT305110), respectively. Based on above results, WG-1 and HrmY-9 was identified as F. oxysporum and F. solani, respectively. Pathogenicity test were performed on healthy one-year-old trifoliate orange seedlings by dipping their injured roots into conidial suspension (50 ml, 1×106 conidia/mL) for 1 h and the rest of conidial suspension was added to the pot after replanting to make sure the inoculum was in contact with the roots. Roots of control plants were inoculated with sterilized water. All experiments were repeated twice. All plants were cultured at 26°C under a 16-h light/dark cycle. Typical symptoms developed on most of inoculated seedlings two months post inoculation. No disease symptoms appeared on control plants. Same colonies were reisolated from the inoculated roots, confirming Koch's postulates. To our knowledge, this is the first report of F. oxysporum and F. solani causing root rot on trifoliate orange rootstock in China. The identification of F. oxysporum and F. solani as the causal agents of the observed root rot on trifoliate orange rootstock is critical to the prevention and control of this disease in the future.

Optimization of Orange Pigment Yield Produced by Monascus ruber under Submerged Fermentation Conditions

Optimization of Orange Pigment Yield Produced by Monascus ruber under Submerged Fermentation Conditions

Accumulation of azafrin in the root apoplast of the medicinal plant Escobedia grandiflora might play a role in parasitism

Societal Impact StatementEscobedia grandiflora is a medicinal hemiparasite that occurs naturally in non‐forested communities in Central and South America. Parasitic plants accumulate high levels of a water‐soluble orange pigment in roots that was once among the most important food dyes in the Andean region. We conclusively address the chemical identification of this pigment and provide molecular and cellular insights on its biosynthesis and possible function.Summary The herbaceous hemiparasite Escobedia grandiflora (Orobanchaceae) is used in traditional medicine in the Andean region. Escobedia roots accumulate high levels of an orange pigment with a significant relevance as a cooking dye that exhibits antioxidant and cardioprotective properties. Here, we aimed to confirm the chemical identity of the pigment and investigate its biosynthesis and function in Escobedia roots. We combined metabolic and cytological analyses with de novo transcriptome assembly, gene expression studies, and phylogenetic analyses. The pigment was conclusively shown to be azafrin, an apocarotenoid likely derived from the cleavage of β‐carotene. RNA‐seq supported by multispecies comparative transcriptome analysis and qRT‐PCR allowed to propose candidate genes for the production of azafrin in Escobedia roots. We also showed that azafrin is delivered to the root apoplast and that it accumulates in the area where the Escobedia haustorium contacts the host's root. Our data suggest that azafrin production might rely on a carotenoid cleavage dioxygenase (CCD) different from CCD7 and that this apocarotenoid might function in the parasitism process. Together, our work represents an unprecedented step forward in our understanding of the Escobedia parasitization system.

Inhibitory effect of Monascus purpureus pigment extracts against fungi and mechanism of action

The fungus Monascus produces several secondary metabolites of different pigment hues. These pigments have shown various biological activities. In this study, Monascus purpureus pigment extracts were tested (in vitro) against Penicillium expansum MTCC 4900, Rhizopus stolinfer MTCC 10595, and Aspergillus niger MTCC 8652 for antifungal activity. The UV–visible spectrum of M. purpureus fermented rice extracts showed λmax at 395, 425, and 500 nm. This indicated the solubility of yellow, orange, and red pigments in polar-based solvent extraction. The M. purpureus pigment extracts inhibited the radial growth and conidial germination of the test fungi. The fungi treated with pigment extract stained with DiBAC (a vital stain) emitted green fluorescence under a fluorescent microscope. These results indicated that the pigment extracts have affected the membrane potential of the treated fungi. Hence, the fungicidal activity of the pigment extracts is due to the disruption of the cell membrane. The HPLC analysis of the pigment revealed the presence of two major peaks. The UV–visible spectrum corresponding to the HPLC peak at 12-min retention time revealed the presence of orange pigment rubropunctatin. Apparently the rubropunctatin present in the extracts exhibited fungicidal activity. Further studies are warranted to assess the applications of M. purpureus pigments in preventing and treating fungus-related diseases.

Supplementary effect of whey components on the monascin productivity of Monascus sp.

Monascus sp. has been used in fermented foods for centuries. It can synthesize yellow, red, and orange pigments as secondary metabolites. Here, we focused on yellow pigment monascin, responsible for anti-inflammation and antidiabetic effects, and investigated whether whey could be a suitable substrate with or without rice powder for monascin production using M. purpureus AHU 9085, M. pilosus NBRC 4520 and M. ruber NBRC 32318. The growth and monascin production of the three Monascus strains were dependent on three liquid media consisting of whey and/or rice. All strains showed the best growth in a rice and whey mixed medium, in which M. ruber NBRC 32318 exhibited the highest total monascin production. Subsequent investigation of the effects of whey components indicated that a mineral cocktail in whey was particularly effective in stimulating the monascin production efficiency of M. ruber NBRC 32318. However, this recipe exhibited less stimulation, or even inhibition, for M. pilosus NBRC 4520 and M. purpureus AHU 9085, respectively. In terms of total monascin production, rice with whey provided the highest amount due to growth promotion along with relatively high production efficiency. The effect of whey on growth and monascin production was strongly dependent on the Monascus strains. Even a mineral cocktail in whey could regulate monascin productivity in a strain-specific manner. Further studies are needed to elucidate the mechanism behind the diverse responses by the minerals in the production of monascin from Monascus. © 2023 Society of Chemical Industry.

Two new species of Astrothelium from Sud Yungas in Bolivia and the first discovery of vegetative propagules in the family Trypetheliaceae (lichen-forming Dothideomycetes, Ascomycota).

Two new species of Astrothelium are described from the Yungas forest in Bolivian Andes. Astrotheliumchulumanense is characterised by pseudostromata concolorous with the thallus, perithecia immersed for the most part, with the upper portion elevated above the thallus and covered, except the tops, with orange pigment, apical and fused ostioles, the absence of lichexanthone (but thallus UV+ orange-yellow), clear hamathecium, 8-spored asci and amyloid, large, muriform ascospores with median septa. Astrotheliumisidiatum is known only in a sterile state and produces isidia that develop in groups on areoles, but easily break off to reveal a medulla that resembles soralia. Both species, according to the two-locus phylogeny, belong to Astrothelium s.str. The production of isidia is reported from the genus Astrothelium and the family Trypetheliaceae for the first time.

First Report of Chestnut Blight Caused by Cryphonectria parasitica on Chestnut (Castanea seguinii) in Gansu Province, China.

In China, chestnut blight usually causes insignificant damage to fruit production of Chinese chestnut (Castanea mollissima Blume) and no serious disease epidemics occur, due to the high resistance to Cryphonectria parasitica (Huang et al. 1998). According to recent surveys, chestnut blight was mainly found in sixteen provinces including Shandong, Hebei, Anhui, Hunan, Jiangxi, Beijing, and Fujian, with severe cases occurring occasionally (Guo et al. 2005). The disease incidence has been aggravated with increasing monoculture of newly improved chestnut cultivars in chestnut-producing areas (Yan et al. 2007), though it was not detected in Gansu Province. In September 2021, some chestnut trees (Castanea seguinii) showing symptoms of crown dieback and diffuse sunken cankers on the trunk with swelled margins and subsequent cracking of the outer bark, were collected in mountains of Hui County in Longnan City, Gansu Province (E 104° 15' 5.76″ ，N 35° 11' 30.84″). Symptomatic branches were washed using tap water and dried on sterilized tissue paper. The Junction between diseased and healthy tissue was cut from the bark and sterilized with NaClO (2.5 %) for 2 minutes, then plated on potato dextrose agar (PDA) and incubated at 25 ℃ for 3 to 4 days. After fungal colonies formed, mycelia were transferred and subcultured onto new PDA media and then purified using single spore culture. After 7 days, colonies turned yellow white. Uninucleate conidia were formed in orange pycnidia and the orange pigments could turn purple if in 2% KOH. Conidia were straight or slightly curved, hyaline, with 2.5-3.5 × 1.2-1.5 μm in size. The characteristics of the culture and morphology were similar with those of C. parasitica (Tziros et al. 2016). Perithecia were not found on culture medium. In accordance with previous findings, the sexual stage of C. parasitica appears on diseased trees in late October. For molecular identification, genomic DNA was extracted from mycelium using a Fungal Genomic DNA Extraction Kit (Tsingke Biotech Co. Ltd, Xi'an, China), the ITS region was amplified with primers ITS1/ITS4 (Sorrentino et al. 2019), and the TEF1-α region was amplified with primers TEF-1H/TEF-2T (O'Donnel et al. 1998). Cloning and sequencing of PCR products were carried out by Tsingke Biotech Co. Ltd, Xi'an, China. The resulting sequences were deposited in GenBank (ITS sequence accession number: OM033734, TEF sequence accession number: OM12254). BLAST results revealed that the sequences of ITS and TEF shared identity over 99% with those of C. parasitica strains (GenBank accession number: AY308953, KP524763, KP824756 and KF220299). Based on morphological and molecular characteristic, the fungal isolates were identified as C. parasitica. To verify pathogenicity, thirty 3-year-old chestnut seeding (70 cm high, 1 cm diameter) of Castanea seguinii were used for inoculation. Chestnut branches were wounded (five wounds per sapling) using a hole punch and inoculated with a mycelial plug (5 mm in diameter) from the edge of 7-day-old, actively growing colonies. Pathogen-free PDA plugs were used as controls. To prevent desiccation, inoculated wounds were sealed with parafilm, and saplings were incubated in a greenhouse at 25℃. Each treatment consisted of 5 seedling and the pathogenicity tests were repeated three times. After inoculation for 5 weeks, symptoms of bark cankers were observed on branches similar to those of diseased chestnut trees in the field. Control saplings with sterile PDA discs did not display symptoms. C. parasitica was reisolated from inoculated branches. To our knowledge, this is the first report of C. parasitica causing chestnut blight in Gansu Province, one of the few areas in the China thought to be free of the disease. The specimens were found in the westernmost part of the natural distribution of chestnuts in China. There are more than 2.6 million chestnut trees, which constitute one of the most important economic forests in Hui County Gansu Province (Yang et al. 2005). The occurrence of chestnut blight could be a restricting factor for chestnut forests.

Mphy gene is involved in temperature-regulated pigment metabolism in Monascus

In plant, Phytochrome (phy) as a light receptor not only plays an important effect on plant morphogenesis, but also is involved in the regulation of plant defense resistance. In Monascus, the formation of conidia, cleistothecia and types and yields of secondary metabolites were found to influence by environmental conditions such as light, temperature, pH, etc. However, the underlying mechanism has not been revealed. Here, the effects of Monascusphy (Mphy) on the regulation of temperature on the growth and development and pigments biosysnthesis of Monascus were investigated by disruption and overexpression of Mphy gene. Colony diameter and number of spores of Mphy knock out strain (ΔMphy) and overexpression strain (OE-Mphy) are reduced compared with the wild strain. The yellow, orange and red pigments yields of original strain was 1.1, 1.2 and 1.1times that of OE-phy strain, and 13.4, 19.3, and 15.2 times that of ΔMphy strain, respectively at 25°C. At 30°C, the yellow, orange and red pigment yields of original strain was 1.3, 1.3 and 1.2 times that of OE-phy strain, and 2.4, 3.7, and 3.0 times that of ΔMphy strain, respectively. However, at 35°C, OE-phy strain had the highest pigment yield, and the pigment yield of the original strain and the ΔMphy strain were very similar. HPLC results showed that the pigment types of primitive strain and OE-phy strain were mainly yellow and orange pigments, and the ΔMphy strain was mainly yellow pigments at 25°C. At 30°C, the original strain had more red, yellow and orange pigments, the OE-phy strain was mainly red and yellow pigments, and the ΔMphy strain was mainly yellow pigments. At 35°C, red and yellow pigments were predominant in the original strain and OE-phy strain, while yellow pigments were predominant in ΔMphy strain.

INVESTIGATING THE MICROBIOTA OF SOLAR SALTERN OF MULUND, MUMBAI, INDIA

Solar salterns, located in tropical and subtropical areas worldwide, are artificial shallow ponds for the production ofhalite (NaCl) from seawater. They are often built as multipond systems. Multi-pond solar salterns have been widespreadenvironments for studies on halophilic microorganisms. In the present study Archaeal and bacterial diversity of thesalterns located at Mulund (19°10’12’’N, 72°57’18’’E), Mumbai was investigated. Three Halophiles were isolated fromthe brine samples collected. All isolated archaea were shown intensely orange pigmentation. These salt tolerantorganisms were characterized using morphological, biochemical and 16S rRNA analysis. Phylogenetic analysis of thesesequences revealed a range of identities to several group of bacteria. The clones fell into major lineages theActinobacteria and firmicutes. Presence of industrially important carotenoids producing halophiles were also recorded.

Nitrogen Enriched Solid-State Cultivation for the Overproduction of Azaphilone Red Pigments by Penicillium sclerotiorum SNB-CN111

Azaphilones are microbial specialized metabolites employed as yellow, orange, red or purple pigments. In particular, yellow azaphilones react spontaneously with functionalized nitrogen groups, leading to red azaphilones. In this study, a new two-step solid-state cultivation process to produce specific red azaphilones pigments was implemented, and their chemical diversity was explored based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and a molecular network. This two-step procedure first implies a cellophane membrane allowing accumulating yellow and orange azaphilones from a Penicillium sclerotiorum SNB-CN111 strain, and second involves the incorporation of the desired functionalized nitrogen by shifting the culture medium. The potential of this solid-state cultivation method was finally demonstrated by overproducing an azaphilone with a propargylamine side chain, representing 16% of the metabolic crude extract mass.

Scale-Up of Pigment Production by the Marine-Derived Filamentous Fungus, Talaromyces albobiverticillius 30548, from Shake Flask to Stirred Bioreactor

Talaromyces albobiverticillius 30548, a marine-derived fungus, produces Monascus-like azaphilone red/orange pigments which have the potential for various industrial applications. The objective of this study was to scale up pigment production in a 2 L bioreactor with a working volume of 1.3 L media and to compare its biomass growth and pigment production against small volume (500 mL) shake flasks with 200 mL working volume. Additionally, fungal morphology, pigment intensity, fermentation length and duration of pigment production were also compared. Experiments were carried out at laboratory scale in 200 mL shake flasks without controlling pH and oxygen. In parallel, fermentation was performed in a 2 L bioreactor as an initial scale-up to investigate the influence of dissolved oxygen, agitation speed and controlled pH on pigment production and biomass growth of T. albobiverticillius 30548. The highest orange and red pigment production in bioreactor at 24 °C was noticed after 160 h of fermentation (70% pO2) with 25.95 AU 470 nm for orange pigments and 22.79 AU 500 nm for red pigments, at pH set point 5.0. Meanwhile, the fermentation using 200 mL shake flasks effectively produced orange pigments with 22.39 AU 470 nm and red pigments with 14.84 AU 500 nm at 192 h under the same experimental conditions (24 °C, pH 5.0, 150 rpm). Regarding fungal morphology, growth of fungus in the bioreactor was in the form of pellets, whereas in the shake flasks it grew in the form of filaments. From the observed differences in shake flasks and closed bioreactor, it is known that the bioprocess was significantly influenced by dissolved oxygen saturation and agitation speed in scale-up. Thus, oxygen transfer appears to be the rate-limiting factor, which highly influences overall growth and production of pigments in Talaromyces albobiverticillius 30548 liquid culture.

First Report of Fusarium acuminatum Causing Leaf Blight on Garlic in China.

In June 2021, leaf blight symptoms were detected on garlic plants (Allium sativum) in southeastern Jiangsu (Nantong municipality; 120.61° E, 33.25° N) in China. Two-month-old garlic plants exhibited leaf tip die back and light brown lesions in new and old leaves (Figure 1). The symptoms were observed in 40% of the plants in a 60-square-meters commercial field surrounded by rice fields, and were similar to those reported for Botrytis porri, Septoria allii and Stemphylium eturmiunum causing leaf blight on garlic (Dumin et al. 2021; Park et al. 2013; Zhang et al. 2009). Six samples of symptomatic tissue collected in Nantong municipality, approximately 1 cm2 in size, were sterilized in 2% NaOCl for 15 min and washed twice with sterile ddH2O. The pathogen was isolated from all collected samples on PDA medium, containing 50 µg/mL chloramphenicol, at 26°C. Pink colonies with orange pigmentation were observed after 7 days. Internal transcribed spacer (ITS), elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes were amplified using ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5 and fRPB2-7CF/fRPB2-11aR primers, respectively. A total of 17 isolates were obtained, with nine of the isolates sharing the same sequences (strain NJC21), six of the isolates sharing the same sequences (strain NJC22), and the other two isolates showing different sequences (strains NJC23 and NJC24). The obtained sequences were submitted in GenBank under accession numbers OL655398-OL655401 (ITS), and OL741712-OL741723 (EF1-α, RPB1, RPB2). The obtained ITS sequences shared >99% homology to the ITS gene from F. acuminatum IBE000006 (EF531232), the EF1-α sequences shared 99% homology to the EF1-α gene from F. acuminatum F1514 (LC469785), the RPB1 sequences shared >99% homology to the RPB1 gene from F. acuminatum JW 289003 (MZ921675), and the RPB2 sequences shared 100% homology to the RPB2 gene from F. acuminatum NL19-077002 (MZ921813) or 100% homology to the RPB2 gene from F. acuminatum MD1 (MW164629). A phylogenetic tree was constructed using MEGA7 with related Fusarium strains (Figure 2). Microscope observations after incubation in potato-sucrose-agar (PSA) medium showed the presence of oval microconidia, fusiform macroconidia, septate mycelium and chlamydospores, and agree with the morphology of F. acuminatum (Marek et al. 2013). The pathogenicity was screened with two-week-old wounded and non-wounded garlic plants using a 1 × 106 spores/mL solution (20 µL). Sterile ddH2O was used in the control experiment. The inoculated plants were incubated at 26°C and 60% relative humidity for 3 days, detecting similar lesions compared to those observed in the field. The pathogen was recovered from 5 different lesions, from different plants, and its identity was confirmed by sequence analysis. Recently, F. acuminatum was reported to cause garlic bulb rot in Serbia (Ignjatov et al. 2017). Although F. acuminatum is well known as a causal agent of root rot (Li et al. 2021; Tang et al. 2021), F. acuminatum has also been found causing leaf blight on onion (Parkunan et al. 2013) and muskmelon (Yu et al. 2021). This is the first report of F. acuminatum causing leaf blight on garlic, demonstrating the host and tissue promiscuity of this pathogen. China is the largest producer of garlic in the world with nearly 20 million tons harvested in 2020. This report will help to better understand the pathogens that are affecting garlic production in China.

Fungal Azaphilone Pigments as Promising Natural Colorants

Abstract—Microscopic fungi form and excrete numerous and diverse secondary metabolites, including pigments of various colors, which may be used as an alternative to chemical and plant colorants used in industry. Azaphilone compounds, first discovered in fungi of the genus Monascus, are among the promising classes of fungal pigments. The review analyzes the publications on formation of azaphilone-type pigments in Monascus fungi, as well as in Talaromyces and Aspergillus сavernicola. Brief information is provided concerning the antimicrobial, antitumor, anti-inflammatory, and hypolipidemic activities of azaphilone pigments. is given. Possible strategies for increasing the efficiency of the production process and directed synthesis of yellow, orange, and red pigments and their derivatives are discussed. In general, the review provides for assessment of the role of azaphilone pigments, as well as of the prospects and ways to expand their production for use as natural dyes in various fields.

First Report of Colletotrichum fioriniae Causing Grapevine Anthracnose in New York.

Grapevine is one of the most widely-planted fruit crops in the world, and is the most economically important fruit crop in the state of New York, USA. Symptoms of anthracnose on grapevine are similarly widely-reported on grapevine fruit and foliage, and such symptoms are commonly attributed to Elsinöe ampelina (Wilcox et al., 2015). However, similar symptoms, if not identical, to those associated with E. ampelina have been sporadically attributed to various species in the genus Colletotrichum. In September 2021, a survey was conducted in three research vineyards at Cornell AgriTech in Geneva, NY. Symptoms of anthracnose werebserved on four Vitis interspecific hybrid breeding lines in a 1 ha vineyard. Leaves, fruit, and petioles showing symptoms of anthracnose, i.e., sunken necrotic lesions with grayish centers and brownish margins, were collected. Symptomatic and healthy portions of surface-sterilized tissues were placed on PDA medium and incubated at 23oC for 7 days. Several petiole samples yielded colonies of white to greyish mycelium, with some red to orange pigmentation (Fig. 1A and 1B), similar to those described by Chowdappa et al. (2009) for Colletotrichum species isolated from grapevine in India. Cultures were allowed to sporulate. Slides from cultures were prepared and examined at 400X magnification. Conidia from cultures were cylindrical with rounded ends, 13.5-15.2 μm in length and 7.6-9.0 μm in width (Fig. 1C). Koch's postulates were fulfilled by inoculating detached healthy leaves of V. vinifera 'Chardonnay' that had been surface sterilized in 10% sodium hypochlorite and triple-rinsed in sterile distilled water. Drop inoculation was used from a suspension of 105 conidia/ml from the foregoing pure cultures as five 2 µL droplets per leaf. Inoculated detached leaves were maintained on water agar in a Petri dish at 23oC. Four days after inoculation, symptoms were observed and compared with the originally collected samples. Inoculated leaves displayed symptoms typically found on the collected tissues, and the original pathogen, as confirmed by colony morphology and conidial characteristics and dimensions, was reisolated from inoculated leaves, and not from non-inoculated controls. For molecular characterization, fungal DNA was isolated by using Qiagen DNeasy kit and amplified using the following primer pairs: ITS1/ITS4, TEF (Hyun et al., 2009), E. ampelina F/R (Santos et al. 2018), TUB2, ACT, HIS3, GAPDH and CHS1 (Damm et al., 2001). PCR products were purified using ExoSAP-IT, and samples were Sanger sequenced. Sequences were analyzed using Geneious Prime software, and the resulting sequences (NCBI accessions OL720215, OL720216, OL720217, OL720218, OL853836, OM982612, OM982613, OM982614, OM982615 and OM982616) had 94 to 100% identity to Colletotrichum fioriniae NCBI accessions MN944922.1, MK646015.1, MN944922.1, MN856415.1, KU847413.1, MN520490.1, MN544294.1, KY695259.1, MN535117.1 and MN544295.1. Symptoms of grapevine anthracnose caused by Colletotrichum species have been reported from India (Chowdappa et al., 2009) and Korea (Kim et al., 2021). To our knowledge this is the first report of grapevine anthracnose caused by C. fioriniae Anthracnose and ripe rot are diseases of increasing importance, particularly as new grapevine cultivars with resistance to powdery mildew or downy mildew are adopted. Taxonomy of the causal agents (E. ampelina and Colletotrichum spp.) has undergone considerable revision. Consequently, distribution and relative prevalence of the various taxa will require further study.

Fusarium pseudocircinatum causing stunting and malformation of sunflower plants in Brazil.

Sunflower (Helianthus annuus L.) is among the main oleaginous crops used in Brazil. During January, 2017, at CCA/UFPB laboratory and greenhouses (Areia/Brazil, 6°58'12″ S; 35°42'15″ W), we observed various sunflower seeds (cultivar Olisun 3, 2017-2018 crop) highly infested with Fusarium. Those seeds were from crops in the municipality of Alagoinha -PB/Brazil (06º57'00'' S; 35º32'42'' W), supplied by Empresa Brasileira de Pesquisa Agropecuária/EMBRAPA. The emerged seedlings from these seeds were also contaminated, with 5% to 26% of them exhibiting stunting and malformation. Fusarium strains were isolated from symptomatic plants, and a single spore was used to grow pure colonies on potato-dextrose-agar (PDA) and synthetic-nutrient-poor-agar (SNA) media. Mycelia of PDA colonies were floccous and dense varying from yellow to orange. Fungal colonies developed aerial mycelium, producing orange pigments. On SNA, hyaline macroconidia, measuring 2.9-4.1 x 32.4-65.0 μm, slightly falcate with three to six septa. Oval microconidia, measuring 2.4-3.6 x 5.1-9.0 μm, were abundant in false heads forming on monophyalides. Chlamydospores were absent. Sterile hyphae were rarely formed. Colectively, the morphological features corresponded to species that belong to the Fusarium fujikuroi species complex (Leslie & Summerell, 2006). To assure the species identity, we sequenced the elongation factor 1α region of two representative isolates (i.e., F2 and F3, GenBank access numbers: MZ666934 and MZ666935, respectively) and compared them to the other Fusarium species found at Fusarium-ID and GenBank databases. Subsequently, we performed a maximum likelihood phylogenetic analysis including previously published sequences (Nicolli et al., 2020). Both isolates exhibited 100% similarity with Fusarium pseudocircinatum (MN386745), and clustered with its ex-type at 100% bootstrap values. The isolates were then grown on PDA amended with manitol to adjust the osmotic pressure to -1.0 Mpa, at 25 ± 2 ° C, for seven days (Sousa et al., 2008). A total of 100 disinfested sunflower seeds (cultivar Olisun 3, 2018-2019 crop) were distributed over the colonies and 48h later they were sown on sterile substrate maintained inside a greenhouse. About 30 days after inoculation, the emerged plants exhibited symptoms of stunting and malformation (60%) compared to controls, which were healthy. F. pseudocircinatum was reisolated from the symptomatic plants, completing Koch's postulates and identified based on above morphological and molecular biological methods. This test was performed twice. Fusarium pseudocircinatum is a broadly distributed and ecologicaly diverse species that infects several wild and cultivated plants. For instance, it was reported on seeds of the wild 'Peroba Rosa' (Aspidosperma polyneuron Muell. Arg.) in Brazil (Mazarotto et al. 2020). Infection of sunflowers may cause plant stand failures, thus resulting in yield and economic losses for Brazilian growers. The correct identification of any pathogen, especialy a generalist one such as F. pseudocircinatum, is crucial to develop eficient management strategies. To our best knowledge, this is the first report of F. pseudocircinatum causing stunting and malformation of sunflower plants in Brazil.

First report of Fusarium equiseti causing leaf spots of Bitter gourd (Momordica charantia) in Pakistan.

Bitter gourd (Momordica charantia L.) is an important vegetable crop of the Cucurbitaceae family widely cultivated in Pakistan and around the world. In October 2020, a nutrition management trial of Bitter gourd cv. Seminis-200) was conducted on an area of 10,860 sq. ft. (99×110 feet) at the Agricultural Research farm of Bahauddin Zakariya University, Multan (30.2601° N, 71.5158° E), Pakistan. Symptoms of large, brown necrotic leaf spots were observed on the leaves of bitter gourd vines. The disease started from the yellowing of leaves within the reticulate venation and turned brown. Irregular brown leaf spots coalesced to form large necrotic areas followed by foliar chlorosis then wilting that occurred very late. There were no crown rot symptoms although there was slight discoloration of roots and when cut longitudinally, browning of tissues was observed. The disease was assessed visually with 37% incidence which resulted in poor quality and yield in terms of reduced size and yellowing of fruit. Infected vines along with the roots were collected for the isolation of pathogen. A total of 34 leaves and 22 root samples were collected from the field for isolation. The leaf, collar and root portions were cut into 0.5 to 1 cm in length and surface disinfected with 1% sodium hypochlorite (NaOCl) for 2-3 minutes followed by washing twice with autoclaved distilled water and after drying, placed on potato dextrose agar (PDA) medium, and incubated at 25±2 °C for one week. The fungal colonies of fluffy white growth with light orange pigment were isolated. For morphological characterization, a total of 4 pure cultures were isolated from leaves, collar region and root by single spore technique on carnation leaf agar (CLA) medium after 15 days of incubation at 25±2℃. Curved and thick-walled macroconidia with elongated or pointed apical characteristic foot-shaped basal cells were produced in sporodochia. Macroconidia with 5-7 septa measured 22.50-41.80 μm × 2.90-4.20 μm (n = 60). Thick, brown with roughened walls and subglobose ellipsoidal chlamydospores were observed in clumps or chains with the dimension of 5.8 to 10.8 μm (n = 20). On morphological characteristics, the fungus was identified as Fusarium equiseti (Corda) Sacc. according to Leslie and Summerell (2006). Two single spore isolates were used for molecular identification by amplifying ribosomal DNA of the internal transcribed spacer (ITS) region with ITS1/ITS4 primers (White et al. 1990) and for β-tubulin gene region, primers T1/Bt-2b (O'Donnell and Cigelnik, 1997) were used. The obtained sequences were deposited in GenBank with accession numbers MW880179 and MW880198 from the ITS region and BLAST search in GenBank showed 100 and 98.11% alignment with previously published sequences of F. equiseti with accessions OM992323.1and MT558569.1 respectively. Accession number OM867571from the β-tubulin region showed 100% sequence similarity with F. equiseti with accession MN653163.1. For pathogenicity, macroconidia from 2-week-old cultures on CLA medium were harvested to prepare spore suspension (1 × 106 conidia/ml). Koch's postulates were confirmed on nine bitter gourd plants (cv. Seminis-200) by applying spore suspension of fungal inoculum at 3-4 leaf stage separately on leaves by automizer, on collar region after making incision spore suspension was applied and in the root zone, 20ml spore suspension was added whereas distilled water was used as a control with three replications. Plants were kept under controlled conditions in the greenhouse with 65% to 75% humidity and the temperature was maintained at 32±2 °C for one week. After 7-8 days, inoculated plants began to exhibit symptoms of brown, necrotic leaf spots on the leaves of bitter gourd vines followed by yellowing of leaves that eventually turned brown. Roots showed slight discoloration and browning of vascular bundles and finally, the plants wilted after four weeks. while control plants remained symptomless. The symptoms resembled those noticed in the field. The fungus was re-isolated from leaves, collar region and roots, followed by morphological identification, and finally confirmed as F. equiseti. To the best of our knowledge, this is the first report of a leaf spot caused by F. equiseti in a bitter gourd from Pakistan. If the disease is not managed properly, it may cause a drastic effect on yield under favorable environmental conditions. The pathogen may also damage other cucurbitaceous crops cultivated in the area.

Phylogeny of the Pyrenula ochraceoflava group (Pyrenulaceae) reveals near-cryptic diversification and the inclusion of the Mazaediothecium album aggregate

In this study we present an analysis of the Pyrenula ochraceoflava group (Pyrenulaceae), focusing on the Neotropics and based on morphological, chemical, and molecular data of the mtSSU, nuLSU and ITS markers. We described three new species from tropical dry forests of Mexico, confirm the monophyly of the P. ochraceoflava group and provide evidence for the inclusion of species currently placed in the genus Mazaediothecium within Pyrenula. Pyrenula aurantiacoretis sp. nov. is characterized by an orange pigment covering the thallus in net-like fashion, muriform ascospores with 4 rows of 1–4 cells each, 12–15.5 × 8–10.5 µm, and 7-chloroemodin and emodin as major compounds. Pyrenula connexa sp. nov. is closely related to Mazaediothecium album, being characterized by mazaedioid pyrenocarps, basal and lateral excipular carbonization, highly variable mature ascospores, 1-septate to submuriform, thallus with abundant white verrucae, and lichexanthone as major compound. Pyrenula moldenkeorum sp. nov. is characterized by an orange thallus, submuriform ascospores that frequently show pigmented septa forming a cross septation pattern, 7.5–11 × 5.5–8.5 µm in size, and 7-chloroemodin and emodin as major compounds. The taxonomy of the most common and widespread species of the group, P. ochraceoflava and P. ochraceoflavens, is briefly discussed, presenting evidence to support the consideration of P. ochraceoflava as a species complex. The two species Mazaedothecium album and M. mohamedii are transferred to Pyrenula as P. aptrootiana nom. nov. [non Pyrenula alba (Schrad.) A.Massal.] and P. mohamedii comb. nov.