Unraveling aerobic cultivable cellulolytic microorganisms within the gastrointestinal tract of sheep (Ovis aries) and their evaluation for cellulose biodegradation

Anaerobic cellulolytic microbes in gastrointestinal tract (GT) of ruminants have been well-documented, however, knowledge of aerobic microbes with cellulolytic activities in ruminant GT is comparably limited. Here, we unraveled aerobic cultivable cellulolytic microbes in GT of Ujimqin sheep (Ovis aries) and evaluated the cellulolytic potential of promising isolates. Twenty-two strains were found to possess cellulose degrading potential by Congo-red staining and phylogenetic analysis of the 16S rDNA/ITS sequence revealed that all strains belonged to nine genera, i.e., Bacillus, Streptomyces, Pseudomonas, Lactobacillus, Brachybacterium, Sanguibacter, Rhizobium, Fusarium, and Aspergillus. Strains with high cellulolytic activities were selected to further evaluate the various enzyme activities on lignocellulosic alfalfa hay (Medicago sativa). Among them, isolate Bacillus subtilis RE2510 showed the highest potential of cellulose degradation considering the high endoglucanase (0.1478 ± 0.0014 IU ml-1), exoglucanase (0.1735 ± 0.0012 IU ml-1) and β-glucosidase (0.3817 ± 0.0031 IU ml-1) after 10-day incubation with alfalfa hay. A significant destruction effect of the cellulose structure and the attachment of B. subtilis RE2510 to the hay were also revealed by using scanning electron microscope. This study expands our knowledge of aerobic cellulolytic isolates from GT of sheep and also highlights their potential application as microbial additive in the aerobic process of cellulose bioconversion.

First Report of Penicillium digitatum causing Postharvest rot of Citron Fruit in Kunming, China

Citron (Citrus medica L.) is a perennial evergreen woody tree of Rutaceae family and Genus of Citrus. The citron is cultivated for its economic, medicinal and ornamental values in the south of China. (Yang et al., 2015). The shapes range from spherical to ovate and the sizes range from 3 to 5 kg (Klein et al., 2016). In June 2021, some postharvest citron fruits (Citrus medica var. medica) were found to have decay with a green or greyish mycelium on part or whole citron in 2 farmer’s markets in Kunming city, Yunnan Province (N 25°02′; E 102°42′), southwest China. Initial symptoms appeared as white, brown, and irregular necrotic spots in the pericarp. The lesions enlarged gradually and developed into green, water-soaked areas which extend rapidly. Eventually, the diseased fruits were rotten, soften, and the green spore masses confined to the surface (Fig. 1A). The incidence of this disease in postharvest citron fruits ranges from 15 % to 35 %, which is extremely destructive to the fruit of Rutaceae family plants (Chen et al., 2019). Small pieces (5 mm2) of symptomatic citron fruits were surface disinfected in 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, rinsed with distilled water for three times, blotted dry, placed onto potato dextrose agar (PDA) medium aseptically and incubated in a growth chamber at 25 ± 1 ℃, after 7 days, different colonies grew on PDA plates that were isolated and purified on new PDA medium at 25 ± 1 ℃ for 7 days. Inoculating repeatedly until six single-strain (XY01 to XY06) were obtained, and these isolates were stored in 15 % glycerol at –80 ℃ in a refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan Agricultural University. The selected pathogens (XY01 to XY06) were inoculated on PDA medium, incubated at 25 ± 1 ℃. After 7 days, colonies of the isolate obverse are olive green, the white margin and greyish-green spores on the surface, and the reverse colorless to cream yellow or pale dull brown. Colonies texture was velutinous, with a special fragrance. The conidia structure was very fragile and break up easily into many cellular elements. Conidiophores were terverticillate, produced by subsurface or aerial hyphae, irregularly branched and composed of short stipes with few metulae and branches that terminate in whorls of three to six phialides, which are often solitary, cylindrical with a short neck. Conidia are hyaline to pale green, smooth-walled, without septate, partially ellipsoidal, or obovate (4.9 to11.9× 4.3 to 8.9 μm). Partial cylindrical (8.2 to 10.5× 2.7 to 5.3 μm), there are some small conidia, which were ellipsoidal or spherical (3.9 to 5.2× 2.7 to 5.2 μm). According to morphological characteristics, the fungus was identified as Penicillium digitatum (Pers.) Sacc. Isolate XY01 and XY02 were used for molecular identification and genomic DNA was extracted using the CTAB method (Aboul-Maaty & Oraby, 2019). The universal primers ITS1 and ITS4 were used to amplify and sequence the ITS1, 5.8S, and ITS2 rDNA region. Using NCBI’s BLASTn tools, the nucleotide sequences of XY01 and XY02 (Gen-Bank accessions MZ976843 and OK513274) show 100 % identity to MK450692 (P. digitatum strain CMV010G4). Pathogenicity tests have used the fruits (Citrus medica), which maturity was more than 80%. The pathogens (XY01, XY02) were cultured for 7 days on PDA medium, washed with sterilized water the resulting spore suspensions diluted to 1.0 × 106 spores/ml. Wounds (0.5 × 0.5 cm) were made on the surface of citron fruits by scraping with a sterile scalpel and then treated with 200 µl of spore suspension (Wild, 1994). Control citron fruits were treated with sterile water. citron fruits were incubated at 24-26 °C. Each treatment was performed in triplicate with 6 citron fruits. After 3 days, all fruits had developed lesions, in a water-stained, pale brown, and rapidly formed white hyphae, white mold layer was observed with a length of 1.5-2.5 cm and a width of 1-2 cm (Fig.1C), but control did induce infection. After 7 days, decay developed more quickly, the hyphae rapidly expanded on the surface of the pericarp, with vague and irregular edges, then a green mold layer was formed, the whole fruit was observed to rot and soften, When the citron was cut, the white flesh inside turned black and rotted (Fig.1B). P. digitatum was consistently reisolated from the inoculated plants but not from the controls. No symptoms developed on the control (Fig.1D). According to Koch’s postulates, the inoculated strains of XY01 and XY02 were the isolates causing citron decay disease. Based on symptoms, morphological characteristics, rDNA-ITS sequence analysis, and pathogenicity, this fungus was identified as P. digitatum. To our knowledge, this is the first report of the distribution of P. digitatum on Citron (Citrus medica) in China.

First report of leaf spot caused by Nigrospora hainanensis on Oxalis corymbosa in China

Oxalis corymbosa DC. introduced into China as an ornamental plant in the mid-19th century is commonly known as an important medicinal and edible perennial herb (Zhou et al. 2021). The plant native to South America is also an invasive and widely distributed weed found in agricultural farms, gardens, and lawns, especially in sugarcane fields of Guangxi province, China. The coverage rate of O. corymbosa in sugarcane fields was normally more than 70%, sometimes up to 100%. In March of 2021, a leaf spot disease of O. corymbosa from sugarcane fields was encountered in Nanning city of Guangxi province, China. Early symptoms appeared as small yellowish round spots. The spots turned to be irregularly, usually exhibiting pale brown necrosis in the center with dark brown necrotic well-defined margins. Severely infected leaves turned to be blighted, then dead. To isolate the pathogen, diseased leave tissue fragments (4 mm × 4 mm) were soaked in 75% ethanol for 10 s followed by 2% sodium hypochlorite for 1 min, and rinsed by sterile water for three times. They were transferred to potato dextrose agar (PDA) medium cultured at 25 °C. Pure cultures were obtained by collected hypha tip from upcoming colonies. The colony features were similar to each other, floccose, white at first, becoming brown, dark brown or black on PDA after 7 days fully covered the 90 mm petri-dishes. Conidial determination were conducted on synthetic nutrient-poor agar medium (SNA) according to Wang et al. (2017). Conidia abundantly dispersed on SNA arising from conidiophores, which normally reduced to conidiogenous cells generated from hyphae. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, 6–8.5 (–12.5) × 5–7.5 (–9) μm in size (n=50). Conidia were solitary, smooth, black, sphaerical or ellipsoidal, (11–) 13–16.5 × (8–) 10–15.5 μm in size (n=100). Setae were not observed during the observation. The fungus was identified as Nigrospora sp. based on the morphology. One of the representative strains (FSC-3) was selected for genomic DNA extraction. The sequences of transcribed spacer region of rDNA (ITS), the partial translation elongation factor (TEF1), and the Beta-tubulin fragment (TUB) were respectively amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F and EF2 (Carbone & Kohn 1999, Crous et al. 2013) and Bt2a and Bt2b (Glass & Donaldson 1995), deposited in the NCBI GenBank with accession numbers of OK083685 (541 bp), OK184809 (481 bp) and OK086377 (421 bp). BLASTn analysis showed that those ITS, TEF1 and TUB gene sequences shared 99%-100% identity with the type strain (CGMCC3.18129) of Nigrospora hainanensis (GenBank accession nos. NR153480, KY019415, KY019464, respectively). In addition, a maximum likelihood analysis using concatenated gene sequences of ITS, TEF1 and TUB was performed in RAxML v.7.2.8 (Stamatakis 2006) implementing the model of GTRCAT with 1,000 bootstrap replicates. The phylogenetic results indicated that the strain FSC-3 was N. hainanensis, which also confirmed after a morphological comparison with N. hainanensis (Wang et al. 2017). Pathogenicity was tested on living Oxalis corymbosa leaves (3 plants for each test) arising from cultivated roots grown for three weeks. It was conducted by dropping 5 μL conidial suspensions (105 conidia / mL) on the living leaves (two sites per leave) incubated in separate containers at 25 °C with 90-100% relative humidity after inoculation. Controls were treated with sterile distilled water. Pale brown small spots came up after 24 h, and then extended to brown larger spots. Symptoms after inoculation were similar to field ones, while the control plants remained healthy. The pathogenicity test was repeated twice with the similar results. Re-isolation of the pathogen from the inoculated leaves was determined based on morphology and sequence analysis to fulfill Koch's postulates. Nigrospora hainanensis had been found from diseased root and leaf tissues of sugarcane in Liuzhou city, Guangxi province (Raza et al. 2019). The results indicated that O. corymbosa was another host in sugarcane fields in Guangxi, China. To our knowledge, this is the first report of Nigrospora hainanensis causing leaf spot on Oxalis corymbosa in China.

First Report of Pulp Rot in the Externally Asymptomatic Pomegranate Fruit Caused by Talaromyces albobiverticillius in Henan Province, China

As a popular deciduous fruit tree, pomegranate (Punica granatum L.) is grown from tropical to temperate zones worldwide, therein China has at least 120000 hm2 cultivation area. In August 2020, severe pulp rot occurred in the externally asymptomatic pre-harvest pomegranate fruit on a 3-year-old soft-seeded variety (Tunisia) in the Zhanghe village (32º40´34˝N, 111º44´20˝E) of Jiuchong township, Xichuan county in Henan province, China with 6.4-20 (av. 12.6) % pulp rot incidence evaluated from 11 freshly sampled fruits (360 pulps per fruit investigated). The fruits showed no external symptoms, however, browning occurred on part of their pulps before harvest compared to the normal ones with white or pink color. The surface of the externally asymptomatic fruits was sterilized with 75% ethanol, and air-dried in a clean bench. The surface-disinfected fruits were dissected with a sterilized knife. Brown pulps from the fruits were picked up using flame-sterilized tweezers and placed on potato dextrose agar (PDA) plates. After five days of incubation at 28 °C, pure fungal cultures with similar phenotypic features developed from the affected pulps. Two randomly selected isolates Tp-2 and Tp-8 were used for the study. The colony surface of the isolates was greyish-green with claret-red exudates. Claret-red pigments were commonly secreted into the medium from the colonies. Conidia were unicellular, hyaline to greyish, mostly rugby ball-shaped with a dimension of 2.2-3.5 (2.7) µm × 1.6-2.0 (1.8) µm (n=50) for Tp-2, and 2.2-3.1 (2.6) µm × 1.6-2.2 (1.8) µm (n=50) for Tp-8. The rDNA internal transcribed spacer (ITS) and β-tubulin gene sequences of the isolates were amplified with primers ITS1/ITS4 and Bt2a/Bt2b, respectively. Sequences were submitted to GenBank with accession numbers MW132153 and MW132077 for the rDNA-ITS sequences, and MW507822 and MW507823 for the β-tubulin gene sequences of Tp-2 and Tp-8, respectively, with a maximal sequence identity greater than 99 % to multiple strains of Talaromyces albobiverticillius (TA) based on BLAST analyses. In the Neighbor-joining phylogenetic trees constructed using rDNA-ITS and β-tubulin gene sequences, both Tp-2 and Tp-8 formed a clade with mutiple strains of TA, clearly separated from other Talaromyces spp. Conidial suspensions (106 spores ml-1) of Tp-2 and Tp-8 were separately injected into five pomegranate fruits (Tunisia) sampled from an orchard free of the disease with a sterilized syringe. Five fruits inoculated with sterilized water were used as control (CK). The inoculated fruits were incubated at 25 °C for 10 days and cut out through the inoculated sites. Pulp rot symptoms occurred in the Tp-2/Tp-8-inoculated fruits, being similar to the naturally affected pulps. The CK pulps remained symptomless during the inoculation tests. Fungal cultures with the same phenotypic features as the inocula were constantly isolated from the brown pulps of the inoculated fruits, verifying both Tp-2 and Tp-8 as the causal agents of the disease based on Koch’s postulates. During a long-term (30-40 days) storage at ambient conditions, fruits sampled from affected orchards developed brown lesions on their peels from which TA cultures could be isolated. TA was reported as the pathogen causing postharvest fruit rot on pomegranate in Italy (Mincuzzi et al. 2017). This is the first report of TA causing pulp rot in the externally asymptomatic pomegranate fruit in China.

Phytophthora Root and Collar Rot of Paulownia, a New Disease for Europe

Paulownia species are fast growing trees native to China, which are being grown in managed plantings in several European countries for the production of wood and biomasses. In 2018, wilting, stunting, leaf yellowing, and collapse, as a consequence of root and crown rot, were observed in around 40% of trees of a 2-year-old planting of Paulownia elongata × P. fortunei in Calabria (Southern Italy). Two species of Phytophthora were consistently recovered from roots, basal stem bark, and rhizosphere soil of symptomatic trees and were identified as Ph. nicotianae and Ph. palmivora on the basis of both morphological characteristics and phylogenetic analysis of rDNA ITS sequences. Koch’s postulates were fulfilled by reproducing the symptoms on potted paulownia saplings transplanted into infested soil or stem-inoculated by wounding. Both Phytophthora species were pathogenic and caused root rot and stem cankers. Even though P. palmivora was the only species recovered from roots of naturally infected plants, in pathogenicity tests through infested soil P. nicotianae was more virulent. This is the first report of Phytophthora root and crown rot of a Paulownia species in Europe. Strategies to prevent this emerging disease include the use of healthy nursery plants, choice of well-drained soils for new plantations, and proper irrigation management.

First report of Anthracnose on Cinnamomun burmannii Caused by Colletotrichum scovillei in China

Mei Pian tree belongs to a new physiological type of Cinnamomun burmannii discovered in the eastern part of the Guangdong province in China in 1987 (Chen et al. 2011). Although the external morphology of Mei Pian tree is similar to Cinnamomun burmannii, the leaves of Mei Pian tree, known as an important traditional Chinese medicine, are rich in natural D-borneol, which protects the heart, brain, and other organs, regulates the central nervous system, and promotes the absorption of other drugs (Yang et al. 2020; Fu et al. 2020). In April 2020, we found that the yield and quality of Mei Pian tree leaves were seriously threatened by anthracnose. Approximately, 40 - 60% of trees were infected in Pingyuan County, Meizhou City, Guangdong Province (N24°28'31.13", E115°50'50.02"). Small circular black spots were initially observed on infected leaves, and spots continued to grow and developed chlorotic margins and concentric rings with sunken areas. As the disease progressed, multiple spots were observed on almost all leaves. Four symptomatic leaves were collected and used for pathogen isolation. The areas of symptomatic and healthy-appearing leaf tissues at the margin of spots were surface-sterilized with 0.5% NaClO for 2 minutes and 70% alcohol for 30 seconds. The sterilized leaves were washed three times with sterile water, air dried, plated on potato dextrose agar (PDA) medium, and incubated at 28°C for 4 days in the dark. A total of six single-spored isolates were obtained and named from MPS-1 to MPS-6, respectively. Among those isolates, MPS-2, MPS-5, and MPS-6 were identical when cultured on PDA plate. The colonies were white to pale gray with dense aerial mycelia, and the reverse side of the colonies was light reddish brown. Conidia were cylindrical and measured 9.0 to 14.0 μm in length and 3.0 to 4.5 μm in width (n = 35). For molecular identification, the primers ITS1/ITS4, GDF/GDR , CHS-79F/CHS-345R, ACT-512F/ACT-783R and T1/Bt2b were used to amplify the partial regions of rDNA-ITS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase(CHS1), actin (ACT) and β-tubulin (TUB2), respectively, from the genomic DNA extracted from fresh mycelia of MPS-2 (Damm et al. 2012). The resulting sequences were deposited in GenBank with accession numbers of MW091490, MW125584, MW125585, MW125586 and MW125587, respectively. The phylogenetic tree was generated by the maximum likelihood method of the MEGA 7 software using a concatenated alignment of ITS, GADPH, CHS1, ACT and TUB2 sequences. According to both morphological and sequence analyses, MPS-2 was identified as Colletotrichum scovillei (Damm et al. 2012, 2020). Pathogenicity tests were performed by inoculating healthy Mei Pian tree leaves with 5 mm PDA plugs containing actively growing mycelium of MPS-2 and wound-inoculated by spraying MPS-2 conidial suspension (106 conidia ml-1). Controls were inoculated only with sterile PDA plugs and ddH2O. All inoculated plants were maintained in a moist chamber (RH greater than 90%) at 25 °C, with an 8-h photoperiod under T5 LED lights. All inoculated leaves developed symptoms similar to those on naturally infected leaves after 5 days, but leaves on control plants remained asymptomatic. The fungus on the inoculated plants was identical in morphology to that found on the original sample collected in the field, thus fulfilling Koch’s postulates. In previous studies, Colletotrichum scovillei also caused anthracnose on banana (Musa spp. AAA group), pepper (Capsicum annuum), and mango (Mangifera indica L.) in China (Zhou et al. 2016; Zhao et al. 2016; Qin et al. 2019). To our knowledge, this is the first report of Colletotrichum scovillei causing anthracnose on Cinnamomun burmannii in China and worldwide. The identification of C. scovillei as the causal agent of the observed anthracnose on C. burmannii is critical to the prevention and control of this disease in the future.

The genetic diversity of angsana (Pterocarpus indicus) in Purwodadi Botanical Garden Indonesia revealed by rDNA ITS

Pterocarpus indicus or angsana is one of the tropical tree species that produce redwood. It has been listed as an endangered species by IUCN since 2018, as its natural population number has declined and possibly extinct in some countries. Therefore, conservation efforts to protect this species must be carried out based on the appropriate conservation strategy. This study aimed to assess molecular characters of Pterocarpus species in Purwodadi Botanical Garden using rDNA ITS (internal transcribed spacer) and its association with morphological characters. Information on genetic and morphological characters will precisely identify this species so that conservation strategy can be appropriately planned. Leaf samples of eighteen P. indicus trees were collected from Purwodadi Botanical Garden (PBG) and used in this study. Twelve specimens that provide clear DNA sequence were genetically assessed. The results showed that P. echinatus exhibited rDNA ITS character similar to that P. indicus, while P. indicus specimens from Morotai were very different from other P. indicus specimens. Morotai specimens showed 89% similarity to several Pterocarpus species, including P. acapulcensis, P. rohrii and P. indicus. The morphological characters were assessed along with molecular characters. The impacts of conservation strategies are discussed in this paper.

Application of Crude Enzymatic Extract In Mushroom Processing To Recover High Value Products

In the present study, a fungal strain was isolated from mushroom waste dump-site and was described based on the morphological and molecular characteristics. The crude enzymatic extract was prepared by fermenting pineapple peels using the newly isolated fungal strain under solid-state condition. The enzymatic saccharification conditions of mushroom were optimized using the central composite design based on the response surface methodology. The isolate had black colony color, conidial head biseriate and small conidia which are synonymous with Aspergillus niger. The phylogenetic analysis using the rDNA ITS sequencing further revealed that the isolate was identical (≥99%) to A. niger. The crude extract displayed CMCase, Fpase and xylanase activities of 20.73U/mL, 34.57U/mL and 118.03U/mL respectively. The saccharification using the crude extract at optimal conditions of pH 6.5, temperature 50oC, enzyme loading of 5% (v/v) and time of 12h achieved maximum glucose yield of 1.639 mg mL-1 which is 1.1 folds higher than the predicted value. This study demonstrated the potential use of crude enzymatic extract from the newly isolated A. niger as a viable and efficient low-cost approach to mushroom processing using enzymes.

Characterization and pathogenicity of Pythium-like species associated with root and collar rot of kiwifruit in Turkey

During the period of June to October in 2018, a widespread decline was observed on kiwifruit vines in the vineyards located in Altınordu, Fatsa, and Perşembe districts of Ordu province. The symptoms were associated with reddish-brown rots expanding from the root to the collar with sparse off-color foliage. Based on the percentage of the total infected samples across 18 vineyards, the most common oomycete species were Globisporangium intermedium (37.1%), Phytopythium vexans (34.3%), G. sylvaticum (14.3%), G. heterothallicum (11.4%), and Pythium dissotocum (2.9%). The morphological identification of isolates was confirmed based on partial DNA sequences containing the nuclear rDNA internal transcribed spacer region (rDNA ITS) and the mitochondrial cytochrome c oxidase subunit II (coxII) gene. The optimum growth temperature and the optimum pH of 5 species ranged from 22.98 to 28.25°C and 5.67 to 8.51, respectively. Pathogenicity tests on the seedlings of kiwifruit cv. Hayward revealed significant differences in virulence among isolates. Phytopythium vexans and G. sylvaticum isolates caused severe root and collar rot resulting in seedling death, while G. heterothallicum and G. intermedium isolates had relatively lower virulence. All Globisporangium spp. and P. vexans isolates significantly decreased plant growth parameters (plant height, shoot and root dry weights and root length); however, P. dissotocum caused very mild symptoms and did not affect these parameters of growth. To our knowledge, this is the first study reporting G. sylvaticum, G. heterothallicum, and G. intermedium causing root and collar rot on kiwifruit not only in Turkey but also in the world.

Studies in the Basidiodendron caesiocinereum complex (Auriculariales, Basidiomycota)

Taxonomy of Basidiodendron caesiocinereum complex is revised based on morphological and molecular methods (with the use of nc LSU rDNA, ITS and TEF1 regions). The basidiospore ornamentation is justified as a key morphological character for the species recognition in the group. As redefined here, B. caesiocinereum is an angiosperm-dwelling species with smooth basidiospores. Bourdotia cinerella and B. cinerella var. trachyspora are proved to represent separate species with warted basidiospores; they are reintroduced as Basidiodendron cinerellum and B. trachysporum. Additionally, eight new species related to B. caesiocinereum are described based on material from Eurasia, North America and Africa, and identity of B. spinosum from Oceania is discussed.