Transparent Polyethylene Bags Research Articles

First Report of Bipolaris zeae Causing Leaf Spot on Setaria viridis in China.

Green bristlegrass (Setaria viridis) is an annual dominant herb in arid sandy grasslands with strong drought resistance to water stress (Valença et al. 2020). In August 2021, brown or dark tan spots on the leaves of S. viridis were observed on natural grassland in Qingyang city, Gansu Province, China (36.55°N, 107.32°E). Leaf disease incidence was around 13%, and the infected area of each leaf was about 20%. For isolation, 30 tissue pieces (5×5 mm) from 10 symptomatic leaf samples were surface-sterilized with 70% ethanol for 30 seconds and rinsed three times with steriled distilled water. Then these tissues were placed on the potato dextrose agar (PDA) at 25 °C, and incubated in dark from 2 to 5 days. Sixteen single-spored cultures with consistent colony characteristics were obtained by single spore isolation as described in Manamgoda et al. (2012). Colonies on PDA were irregular shapes with black color and white edge; conidia were dark brown with septate, cylindrical, straight or flexuous geniculate at the upper end, 22.7 to 69.38×9.2 to 17.6 µm (n = 50; average size 40.8×13.2 µm) under microscope (40X). The internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GPD) regions of the representative isolate 83HXGWC, 84HXGWC and 85HXGWC were amplified using the primers of ITS1/ITS4 and GPD1/GPD2 (Manamgoda et al. 2012). All the amplified gene sequences were deposited into GenBank with accession numbers ITS (OP703331, OQ978850, and OQ978851) and GPD (OQ559683, OQ992505, and OQ992506). BLAST analysis of both the two segments showed 100% identity with those reported sequences of ex-type isolate BRIP 11512 (KJ415538 and KJ415408) of B. zeae Sivan (Tan et al. 2014; Bhunjun et al. 2020), respectively. In the maximum-likelihood phylogenetic tree, 83-HXGWC, 84HXGWC and 85HXGWC strains were grouped with the reference sequences of B. zeae with a high bootstrap supporting values of 100%. Five healthy green bristlegrass (6 weeks) plants in pots were sprayed with conidial suspensions (1×106 conidia ml-1). Another five pots were sprayed with sterilized distilled water (controls). Then all pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse maintained at 18 to 25 °C. After incubation for 14 days, the typical symptoms of leaf spots developed from brown to dark brown on the plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The measurement of pathogenicity was carried out three times. The same pathogens were consistently reisolated from inoculated leaves, and was confirmed as B. zeae based on morphological and molecular analyses, fulfilling Koch's postulates. This study provides a new record of B. zeae on green bristlegrass on natural grassland in China, which can potentially impose disease challenges to other crops. Since, B. oryzae has been known to cause disease in cereal crops (Manamgoda et al. 2014), this can be a potential alternate source of survival of this pathogen.

First Report of Pseudopithomyces palmicola Causing Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely cultivated for the production of forage, hay and silage worldwide, due to its high nutritional value and palatability (Feng et al. 2021). It has been infected by a number of foliar fungal diseases caused by different fungal pathogens (Xue et al. 2017, 2020; Victoria Arellano et al. 2021; Liu et al. 2023). In August 2021, three Pseudopithomyces isolates with similar colony characteristics were isolated from fresh samples of leaf spots on Italian ryegrass that was collected from the Forage Germplasm Nursery, Maming town of Qujing city, Yunnan province, China (25.53833°N, 103.60278°E). For specific isolation, tissue pieces (about 0.5 cm × 1 cm) from symptomatic leaves were surface-disinfested in 75% ethanol solution for 40s, rinsed three times in sterilized distilled water and air dried, plated on potato dextrose agar (PDA), and incubated at 25°C for 3 to 7 days in the dark. Following initial isolation, a representative isolate, KM42, was selected for further study. Colonies growing on PDA were cottony, white to grey, 53.8 to 56.9 mm diameter after 6 days in the dark at 25°C, with white and regular edge; in reverse, colonies were brown to dark brown in the center, and pale yellow toward the periphery with concentric rings. Under near-UV light at room temperature (20 ± 3°C), colonies were incubated on PDA for ten days to produce conidia. Conidia were globose or ellipsoid to amygdaloid, 1- to 3-transversely septate, 0- to 2-vertical septate, light brown to brown, measuring 11.6 to 24.4 × 7.7 to 16.8 µm (avg. 17.3 × 10.9 µm). The internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA(ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified with primers described by Chen et al. (2017). Sequences were deposited in GenBank (OQ875842 for ITS, OQ875844 for LSU, and OQ883943 for RPB2). BLAST analysis of all these three segments showed 100% (ITS: MF804527), 100% (LSU: KU554630), and 99.4% (RPB2: MH249030) identity with sequences of reported isolate CBS 143931 (= UC22) of Pseudopithomyces palmicola (Lorenzini et al. 2016; Liu et al. 2018). To fulfill Koch's postulates, four 12-week-old healthy Italian ryegrass plants were spray-inoculated separately with a mycelial suspension of about 5.4 × 102 CFU/ml of an isolate of P. palmicola. In addition, four control plants were sprayed with sterilized distilled water. All plants were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 22°C. Small brown to dark brown spots developed on leaves 10 days after inoculation; symptoms were not observed on control plants. Pathogenicity tests were conducted three times by the same method. The same fungus was re-isolated from the lesions, and confirmed by morphological and molecular methods as described above. To the best of our knowledge, this is the first report of P. palmicola causing leaf spot on Italian ryegrass in China or worldwide. This information will be useful to forage grass managers and plant pathologists in recognizing the disease and developing effective control measures.

Characteristic of Freshness Ulva lactuca Under Different Temperatures at Short-term Storage

Ulva sp. is green macroalgae that very potential for producing food with high nutrients content. This species is found on a coastline in intertidal zone of Indonesia’s waters. There have been several research to this species start from post-harvest handling, nutrition, probiotic, and its ulvaran, however the information of effect of temperature storage to Ulva lactuca freshness is still lack. This study was undergone to evaluate the freshness quality of Ulva lactuca by sensory changes in different temperatures short-term storage. In order to evaluate the effect of the different storage circumstances, the fresh U. lactuca was collected from its natural habitat (intertidal zone of Sepanjang Coast, Yogyakarta Indonesia). It was rinsed from debris and epiphyte, stored in transparent polyethylene bag, and were stored in 4 °C, 15-20 °C and room temperature for five days. Color, pH, ash, moisture, crude protein, chlorophyl, Total Plate Count, sensory analysis was undergone along the storage period. The sensory evaluation score of U. lactuca in 4 °C were more than 6, and better than other storages. The crude protein (U. lactuca stored in 4 °C) decreased significantly on day 4 by 5.53%, it was lower than others. The TPC of all samples varied from 147x103in 0 days of storage to 2,462.5x103 CFU/ml on the last day of storage. In summaries, sensory scores of U. lactuca are more constant and higher in 4 °C than in other storages, despite minor nutrient content deterioration.

First Report of Didymella sinensis Causing Leaf Blight on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum Lam.) is a high-yield, high-quality forage grass and is cultivated widely in southern China. In April 2021, small black spots were observed on leaves of Italian ryegrass in the field of about 300 ha located in DuShan county, Guizhou province, China (25.62056°N, 107.53139°E). Approximately 1 to 3% of plants were affected. For isolation, eleven tissue pieces (about 0.5 × 1 cm) from four symptomatic leaves were surface-disinfested in 75% ethanol solution for 40s, rinsed thrice in sterilized distilled water and air dried; then these tissues were plated on potato dextrose agar (PDA), and incubated at 25°C for 4 days in the dark. Nine fungal isolates with similar colony characteristics were obtained, and three representative isolates (LMDS1, LMDS2 and LMDS3) were selected for further study. Colonies on PDA were 47 to 57 mm diam after 5 days, margin regular, dark gray in the center surrounded by white to gray, with floccose aerial mycelia on the upper side, and dark brown to black on the reverse side. There was no fungal sporulation when these isolates were incubated under continuous ultraviolet light on PDA, oatmeal agar (OA), malt extract agar (MEA) and potato carrot agar (PCA). ITS-rDNA, LSU-rDNA, and two other protein-coding genes (RPB2 and TUB2) were amplified with primers described by Chen et al. (2017). Sequences were deposited in GenBank (ON692740 to ON692742 for ITS, ON692775 to ON692777 for LSU, ON704660 to ON704662 for RPB2, and ON704657 to ON704659 for TUB2). BLAST analysis of all these four segments showed >99.7% identity with those sequences of ex-type isolate CGMCC 3.18348 of D. sinensis (Chen et al. 2017; Hou et al. 2020). Maximum likelihood (RAxML) phylogenetic tree based on the combined ITS, LSU, RPB2 and TUB2 alignments also showed these three isolates and the other two reported D. sinensis isolates formed a subclade with 100% bootstrap support. Referring to our previous method (Xue et al. 2020), five 8-week-old healthy plants of Italian ryegrass were spray-inoculated separately with a mycelial suspension of about 1.5 × 104 CFU/ml. In addition, five plants considered as non-inoculated controls were sprayed with sterilized distilled water. All plants were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 23 to 26°C. The small black spots similar to those observed on infected plants in the field developed on leaves fifteen days after inoculation. The symptoms consisted of brown to dark brown spots when leaves were severely infected; however, symptoms were not observed on non-inoculated plants (controls). Pathogenicity tests were carried out three times. The same fungus was re-isolated from the lesions, and confirmed by morphological characterization and molecular technique as described above, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of D. sinensis causing leaf blight on Italian ryegrass in China. The accurate identification of this pathogen would be useful for the prevention and control of leaf spot on Italian ryegrass in the future.

First report of leaf blight caused by Pantoea agglomeranson wheat in China.

Wheat (Triticum aestivum L.) is the main grain crop in Ningxia Hui Autonomous Region, China. A new leaf blight disease of wheat was observed in many wheat fields in Yinchuan City and Wuzhong City of Ningxia during 2020-2021. The average disease incidence of the cultivar Ningchun 50 was 5 to 15%, and there appeared the evident disease symptoms from the heading stage, then the symptoms got more serious until the mature stage. The tips of the leaves were chlorotic and turned bright yellow at the early stage of the disease. Later on, the yellow leaf spots were further spread from the tip to the petiole, and the yellow-colored necrotic lesions emerged, resulted in withering and death of leaves (e-Xtra 1, a-d). To isolate and identify the pathogenic agent, diseased leaves were cut into 0.5 cm × 0.5 cm small pieces, and sterilized in 5% NaOCl solution for 5 min, and were rinsed three times in sterile water, then crushed with tweezers in 2 mL sterilized water and streaked three times onto Nutrient Agar (NA) medium. and 15 single colonies which had the same colony morphology were obtained. Of the 15 colonies, 3 (named WH1, Cx1 and HJ1) were randomly selected for further morphological, biochemical and molecular characterization. The resulting bacterial colonies were incubated at 29±1°C in the dark for 3 days, colony morphology was raised, mucoid texture, round, and smooth with entire margin; the color of these colonies was white at the beginning and turned yellow later. These bacteria were rod-shaped gram-negative cells with peritrichous flagella. Based on the physiological and biochemical assay results (e-Xtra 2), the three strains were initially identified as Pantoea agglomerans (Wang, D H., et al. 2021; Wang, J J., et al. 2021). 16S rDNA and gyrB of the three strains were amplified and sequenced by ABI3730XL sequencer in GENEWIZ (Suzhou, China). The sequences of 16S rDNA and gyrB of these strains were submitted to GenBank with the accession numbers ON428446, ON428461 and ON428462 for 16S rDNA; ON461799, ON461801, ON461803 for gyrB. 16S rDNA and gyrB sequences homology analysis showed that the three strains had the highest homology which were over 99.5% with the sequences of the reported P. agglomerans (e-Xtra 1, g) . A phylogenetic analysis based on 16S rDNA and gyrB gene sequences was performed using the MEGA6.0 proximity method, and the results of the phylogenetic tree showed that strains Cx1, WH1 and HJ1 clustered on the same clade with the reported P. agglomerans strains (e-Xtra 1, h-j). Thus, Cx1, WH1 and HJ1 were identified as P. agglomerans. Pathogenicity test was performed to complete Koch's postulates, Ningchun 50 was planted in pots, four-week-old healthy wheat seedlings were inoculated with 107 CFU/mL bacterial suspension using two inoculation methods: 1) Leaf surface was poked with disposable syringe needle, and 50 µL of suspension was injected into each of the pinholes (Suraj, S., et al. 2020); 2) Leaf was cut at 45° at the lower 2-3 cm of the leaf tip with scissors dipped in the bacterial suspension. Wheat leaves inoculated with sterile distilled water were regarded as controls. The inoculated wheat was cultivated in a greenhouse (temperature 28 ± 2°C, humidity 40 ± 2%) and covered in transparent polyethylene bags at first 96 h. Symptoms appeared at 3 days after inoculation, and after 7 days, the acupunctured wheat leaves turned chlorotic and yellow around the pinholes and some were necrotic; the leaf-cutting wheat turned yellow and necrotic from the clipping point to the leaf base; the acupunctured and cut leaves totally died after 15 days, and all of the control leaves were healthy (e-Xtra 1, e-f). Subsequently, pathogens were reisolated from inoculated leaves, and identified as P. agglomerans according to molecular identification described above. To our knowledge, this is the first report of leaf blight disease of wheat caused by Pantoea agglomeransglobally as well as in China. Identifying the cause of the disease will support efforts for its future control and management.

First Report of Brown Leaf Spot of Elymus nutans Caused by Bipolaris sorokiniana in Northwestern China.

Elymus nutans is an important feed and forage resource in alpine region of northwestern China, and helps to maintain the ecological balance on Qinghai-Tibetan plateau (Fei et al. 2010). In September 2021, brown leaf spots were observed in Shandan Horse Farm (38.3061°N, 101.2453°E), Gansu province of China.Disease severity of leaves was estimated to be 38% based on a random sample of 100 leaves from each of 10 arbitrarily placed quadrats on the farm.The typical symptoms on leaves appeared as brown to dark brown spots with grayish white in the center, surrounded by a yellow halo. A total of twenty diseased leaves were collected from five different plants in the field. The lesions (about 5 mm) from these samples were initially soaked in 75% ethanol for 30 s followed by soaking in 1% NaClO solution for 1 min, repeatedly rinsed with sterilized water, air dried, and put on potato dextrose agar (PDA). After 7 days of incubation at 25°C in the dark, the same fungus was consistently isolated; the pure isolates were obtained by single-spore cultures (Cai et al. 2009). The colonies were irregular in shape and black in color with white margins. Conidia were spindle-shaped, dark brown, 5 to 9 septa (7 septa in general), and a size range of 21.74 to 78.97 × 7.95 to 21.12 µm (avg. 57.25 × 17.76 µm, n = 50),morphologically similar to B. sorokiniana reported on Avena nuda in China (Li et al. 2019). The ITS region of rDNA and partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of the representative isolate PJC5 were amplified using the primers ITS1/ITS4 and gpd1/gpd2, respectively (Manamgoda et al. 2014). After sequencing sequences were deposited into GenBank with accession numbers OM419194 (ITS) and OM718009 (GAPDH). BLAST analysis showed 99% and 100% identity with the corresponding sequences (accession numbers MH855159.1 and HG779088.1) of known strain CBS 140.31 of B. sorokiniana (Manamgoda et al. 2014). For pathogenicity tests, ten 8-week-old healthy plants obtained by growing E. nutans in pots were spray-inoculated separately with a conidia suspension of 1 × 106 conidia/ml of isolate PJC5. Ten plants were also sprayed with sterilized distilled water as controls. Then all plants were individually covered with transparent polyethylene bags for three days to maintain high relative humidity and placed in greenhouse at 25°C. Seven days after inoculation, symptoms of leaf infection were similar to those observed in the field, while no symptoms appeared on leaves of control plants. The experiments were conducted three times and the pathogen was re-isolated from inoculated leaves and was confirmed as B. sorokiniana based on morphological and molecular analyses.B. sorokiniana has been reported to cause leaf spot of E. riparius in the United States (Roane et al. 2005) and E. breviaristatus (Zhuang, 2005) and A. nuda (Li et al. 2019) in China. To our knowledge, this is thefirstreportofB.sorokinianacausing leaf spot onE. nutansin China. Identifying this pathogen provides a foundation to prevent this complex disease and to reduce economic loss.

First Report of Curvularia lunata Causing Leaf Spot on Oryza sativa in Sabah, Malaysian Borneo.

Rice (Oryza sativa L.) has been farmed in Malaysia since ancient times and is one of the most important commercial crops (Ma'arup et al. 2020). Throughout January to August 2022, chlorotic spots with brown halos ranging 2 to 10 mm wide were found on upper leaves of rice variety Mahsuri in the vegetative stage with a severity and incidence of approximately 60% and 100%, respectively in Kampung Tagas, Sabah, Malaysian Borneo (06°09'41.8"N, 116°13'45.1"E). As the disease developed, the spots coalesced into larger chlorotic spots. Three leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022a), before plating on water agar (WA) at 25°C. Purification of fungi was conducted on WA using hyphal tip isolation. When three pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) and WA for 7 days in 12 h light and 12 h dark at 25°C for the macro- and micro-morphological characterization, respectively. The colonies of the three isolates on PDA were initially gray, later becoming dark. Conidia (n=30) were fusiform, smooth-walled, dark-brown, and melanized with three transverse septa, measuring 7.3 to 11.4 × 16.2 to 27.2 µm. The isolates were named Tagas01, Tagas02, Tagas03. Genomic DNA was extracted from fresh mycelia of the pathogens based on the extraction method described by Khoo et al. (2022b). The primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1991), and EF1-983F/EF1-2218R (Schochet al. 2009) were used to amplify the internal transcribed spacer (ITS) region of rDNA, partial fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor (EF-1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022a). The sequences were deposited in GenBank under accession numbers OP268402, OP271304, OP271305 (677/677 bp) (ITS), OP270699, OP270703, OP270704 (609/613 bp) (GAPDH), OP270700-OP270702 (928/930 bp) (EF-1α). They were 99.35-100% similar to the Curvularia lunata ITS (HF934911), GAPDH (LT715821), and Curvularia dactyloctenicola EF-1α (MF490858) type sequences. Although C. dactyloctenicolais related to C. lunata, the conidia of the former are much wider making them easier to differentiate (Marin-Felix et al. 2017). Phylogenetic analysis using maximum likelihood based on the combined ITS, GAPDH and EF-1α sequences indicated that the isolate formed a supported clade to C. lunata. The pathogens were identified as C. lunata based on morphological and molecular characterization. Koch's postulates were performed. Three replicate healthy rice at the vegetative stage were sprayed with a spore suspension of 1 × 106 spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on WA. Three replicate rice plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to keep moisture, and kept in a greenhouse at ~27°C. Bags were removed after 4 days of incubation. Monitoring and incubation were performed in greenhouse based on Iftikhar et al. (2022). The pathogenicity test was also performed using isolate Tagas02 and Tagas03. All inoculated leaves developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls. The experiments were repeated twice. The reisolated fungi were identical to the pathogen morphologically and molecularly, thus fulfilling Koch's postulates. C. lunata has been reported in Peninsular Malaysia (Lee et al. 2012). This is the first report of C. lunata causing leaf spot on Oryza sativa in Sabah, Malaysian Borneo. This illness not only reduces yields and lowers milling quality, but it may also be mistaken for rice blast, necessitating needless fungicide spraying.

First report of Curvularia intermedia Boedijn causing leaf blight disease on Microstegium vimineum in China.

Stiltgrass [Microstegium vimineum (Trin.) A. Camus], is an annual C4 grass of Asiatic origin whose native range includes India, Pakistan, Nepal, China, Korea, and Japan (Cole et al 2004). In China, it is mainly distributed south of the Yangtze River, and is one of the most important weeds in autumn-maturing dryland crops, orchards, tea gardens, and plantations. With its high shade tolerance, M. vimineum also invades forest understories and crowds out the local vegetation (Warren et al. 2011). From June to August 2019, a leaf disease was observed causing severe defoliation of stiltgrass on the roadside of Sun Yat-sen Mausoleumin Nanjing City, Jiangsu Province, China (32.045964°N, 118.840064°E). Yellow or yellow-brown necrotic spots were observed on leaf tips and margins of the lower canopy, which later expanded to the entire leaf and progressed up the plant. Disease incidence was approximately 75-85% in August. Thirty symptomatic leaves were collected, and tissue samples (5 × 5 mm) were surface disinfected with 75% ethanol for 30 s, 0.02% NaClO for 30 s, 75% ethanol for 30 s, and washed twice with sterile water. Disinfected tissues were placed on potato dextrose agar (PDA) and incubated at 28°C for 5 days. Twenty-seven morphologically similar isolates were obtained from the leaves and purified by single-spore culturing for further study. Colonies on PDA were 70 to 85 mm in diameter after 4 to 5 days, initially white becoming gray-green with flocculent aerial mycelia. Conidiophores were solitary or clustered, 85 to 139 µm long × 5 to 8 µm wide (n = 50), and conidia were obclavate to ellipsoid or spindle shaped, brown, and measured 28 to 37 µm long × 13 to 18 µm wide (n = 50) with three false dissepiments. All characteristics were consistent with the morphology of Curvularia intermedia Boedijn (Sivanesan 1987). The rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and translation elongation factor (TEF) of a representative isolate (JSNJ-2019) were amplified using primers ITS1/ITS4, GPD1/GPD2 and EF1-983F/EF1-2218R (Manamgoda et al. 2014). The ITS sequence of JSNJ-2019 (GenBank: MZ613310) showed 99.83% (582/583bp) identity with C. intermedia (GenBank: MF370184 and GU073102); the GPDH sequence (GenBank: MZ701795) showed 99.66% (581/583bp) identity with C. intermedia (GenBank: LT715828) and the TEF sequence (Genbank: OM282974) showed 99.77% (864/866bp) identity with C. intermedia (GenBank: MF370186). Phylogenetic analysis based on the TEF sequences using Maximum-Likelihood and Bayesian methods placed JSNJ-2019 in the same clade with reference strain C. intermedia B19. The isolate was deposited in China Centre for Type Culture Collection (CCTCC) (Isolate code: CCTCC AF 2022041). For the pathogenicity assay, ten healthy M. vimineum plants grown in plastic pots (five to six leaf stage) were sprayed with 20ml conidial suspension (5×104 spores /ml); another ten healthy plants sprayed with sterile water served as controls. All inoculated and control plants were covered with transparent polyethylene bags immediately and were maintained in a greenhouse at 28±1℃. The transparent polyethylene bags were removed after 24 hours. The pathogenicity test was repeated three times. Five days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms was observed on control plants. Reisolations were performed from inoculated plants, and the reisolated pathogen was confirmed as C. intermedia inter based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. Host range tests showed, C. intermedia JSNJ-2019 was pathogenic on corn, wheat, sorghum, barnyardgrass, crabgrass, green foxtail, Chinese sprangletop, cynodon, cogongrass, goosegrass, purslane and bedstraw and non-pathogenic on barley, rice, oat, cotton, bean, peanuts, rapeseed, tobacco and tea. These findings suggest C. intermedia could be used as a biocontrol agent against invasive M. vimineum and farmland weeds. However, application of C. intermedia as a bioherbicide should be limited to insensitive crop growing areas.

Effects of packaging and ripeness on plantain flour characteristics during storage.

The increasing implication of plantain flour in various food formulations calls for the need to evaluate the effects of ripening stage, packaging materials, and storage duration on its proximal composition and functional properties. For this study, plantain flours were produced from the cultivar Alloga at unripe and semiripe stage 3. They were stored both in transparent polyethylene bags and in an opaque aluminum foil. Physicochemical analyses and functional characterization of the plantain flour were performed on samples taken prior to storage and on monthly basis for 6 months during storage. Ash and carbohydrate contents decreased while the yellowness and redness increased with ripening. Pasting viscosity drastically decreased with ripening. During storage, significant differences in color and among most functional characteristics were observed as a consequence of both storage duration and packaging materials. Based on this research, flour from semiripe plantain could be recommended for use in formulations requiring low viscosity. Besides, it is suggested to store plantain flours in opaque containers to reduce the variability in its properties, thus maintaining its original quality.

Identification of Fungi Associated with Irvingia gabonensis (Ogbono) Seeds Spoilage in Benin City, Nigeria

Irvingia gabonensis (Ogbono) is one of the forest tree species of great domestic importance and its postharvest shelve life is affected by various species of fungi. This study is to investigate the effect of various species of fungi that attack Irvingia gabonensis seeds in post-harvest. Ready to use cotyledons of Ogbono were obtained from six (6) markets in Benin City (New Benin market, Aduwawa market, Uselu market, Oba market, Ikpoba Hill market, Santana market) Edo State, in sterile transparent polyethylene bags and transported to the laboratory for analyses. The proximate composition of the Ogbono seeds was determined using standard protocols. The pour plate method was used for isolation using a potato Dextrose Agar which was supplemented with streptomycin. The fungi were isolated based on their cultural and microscopic characteristics. The result of the highest and lowest fungal counts were too numerous to count and 3.3 × 103 cfu/g respectively. A total of seven species of fungi were isolated and identified in this study which included Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Mucor sp., Neurospora sp., Penicillium sp., and Rhizopus sp. Aspergillus flavus was the most prevalent fungi with prevalence rate of 48 (90.6%), while Neurospora sp. was the least prevalent fungi with prevalent rate of 04 (18.2%). The presence of these fungi especially Aspergillus species portrays a serious public health implication as regard food poisoning, mycotocosis and food security. Proper handling methods of Ogbono seeds, coupled with good orientation are necessary to ensure the safety of ogbono seeds.

Modified Atmosphere Packaging Maintains the Sensory and Nutritional Qualities of Post-harvest Baby Mustard During Low-Temperature Storage.

Baby mustard is a popular, yet highly perishable, Brassica vegetable. There is a need to develop effective methods for maintaining post-harvest qualities of baby mustard. Here, the lateral buds of baby mustard were packed in transparent polyethylene bags with no holes (M0), 6 mm in diameter holes (M1), or 12 mm in diameter holes (M2) and stored at 4°C. The effect of different modified atmosphere packaging (MAP) treatments on the sensory quality, health-promoting compounds, and antioxidant capacity was investigated by comparison with non-wrapped baby mustard. M1 and M2 delayed sensory quality deterioration and slowed declines in the content of ascorbic acid, total phenolics, and glucosinolates and antioxidant capacity during storage. M1 was most effective in prolonging the shelf life (three additional days compared with control lateral buds) and maintaining the content of glucosinolates. However, M0 accelerated the decline in the odor score, acceptability score, and ascorbic acid content and shortened the shelf life of baby mustard by more than 5 d compared with the control. These findings indicate that the effect of MAP treatment depends on the size of the holes in the bag. Based on these results, M1 was an alternative method for prolonging the shelf life and maintaining post-harvest qualities of baby mustard stored at 4°C.

First Report of Ergot (Claviceps purpurea) on Drunken Horse Grass (Achnatherum inebrians) in China.

Drunken horse grass (Achnatherum inebrians) belongs to the family Poaceae: it is mainly distributed in the natural grasslands of northern and northwestern in China. Ergot is a disease that can not only affect the growth of the grass, but also cause livestock poisoning (Coufal-Majewski et al. 2016). In September 2018, ergot was observed in a large area (about 15 ha) in Xinghai county, Qinghai province, China (35° 47' N, 99° 53' E, Altitude 3559 m). Around 65% of the plants of Achnatherum inebrians were affected. Symptoms initially showed drop-like honeydew on the ears of drunken horse grass, and later brown to dark brown sclerotia were observed. These were straight to slightly curved, measured 6.7 to 13.5 × 1.5 to 2.1 mm, which was approximately 1 to 4 times the size of healthy seeds. Sixteen spikes with typical symptoms were collected from eight different fields. Sclerotia were disinfested by immersion in 75% ethanol for 30 s and 1% NaClO for 90 s, rinsed three times in sterilized water, plated on potato dextrose agar (PDA) medium, incubated at 24°C in the dark and isolates purified by culturing from single spores. Finally, 16 single-spore cultures with similar phenotypes were obtained from these sclerotia. Colonies produced on PDA for 15 days at 24 ℃ were grayish white with fluffy aerial mycelium, about 60 mm in diameter. Conidia were hyaline, ovoid to cylindrical, 5.42 to 7.69 × 2.85 to 3.75 μm (avg. 5.67 × 3.2; n = 50). These morphological characteristics were consistent with the descriptions of Claviceps species in general (PíchovÁ et al. 2018). To further identify the Claviceps spp., isolate NSZJ (=MHLZU-AI20201012) was selected as a representative for molecular characterization. Two nuclear protein-coding genes TUB2 and MCM7 were amplified by T2/T12 (O'Donnell and Cigelnik 1997) and CARCA-F/M456-5R (Rehner and Buckley 2005), respectively, and sequenced. Sequences were deposited in GenBank (accession nos. MW115640 for TUB2 and MW115641 for MCM7). A BLAST analysis of these two segments showed >99% identity with those sequences of isolate W3 of C. purpurea (Pazoutová et al. 2014). To confirm the pathogenicity on drunken horse grass, 20 healthy plants (2-year-old) grown in an experimental field at the College of Pasture Agriculture Science and Technology, Yuzhong Campus of Lanzhou University in China (104° 39' E, 35° 89' N, altitude1653m) were spray-inoculated with conidial suspension (1 × 106 conidia/ml) during the flowering period. Another 20 plants in the field were sprayed with sterilized distilled water as controls. All plants were individually covered with transparent polyethylene bags for 24 h to maintain high relative humidity. After 7 to 10 days, small yellowish-white drops of honeydew were observed in some florets. At 22 days post inoculation, all the inoculated panicles developed three to six sclerotia per head, ranging in size from 6.7 to 13.5 mm, while control plants remained healthy. The same pathogen was consistently re-isolated from inoculated spikes and confirmed by morphological and molecular characterization as described above. Claviceps purpurea was reported to be associated with ergot in A. lemmonii in Idaho and A. robustum in Montana (Alderman et al. 2004). To our knowledge, this is the first report of C. purpurea causing ergot in A. inebrians in China.

The First Report of Leaf Spot Caused by Alternaria alternata on Italian Ryegrass (Lolium multiflorum) in China.

Italian ryegrass (Lolium multiflorum Lam.) is one of the most important forage crops in southwestern China. In 2018, a leaf spot was observed in a field of Italian ryegrass in Mengyang, Sichuan province, China (30.96925°N, 104.10223°E). From January to early March, this leaf spot developed sporadically and appeared as brown to dark brown lesions. In late May, this disease reached a peak with incidence up to 80% and appeared as reddish-brown necrotic spots with a grayish white to brown center. To isolate the pathogen, sections (0.5 × 1 cm2) of 30 diseased leaves collected from 10 plants were surface-disinfested in 70% ethanol solution for 30 s, 5% NaOCl solution for 5 min, rinsed thrice in sterilized distilled water, air dried, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 4 days. To obtain pure isolates, the single-spore isolation technique (Cai et al. 2009) was used. The conidial suspensions were diluted to a reasonable concentration, spread onto PDA, and incubated at 25°C in dark for 24 to 48 h, and then single germinated conidia were transferred onto new PDA plates (Cai et al. 2009). Nine pure isolates showing similar morphology were obtained for further study. Colonies on PDA were dark gray in the center surrounded by white to gray, with gossypine mycelia on the upper side, and red to dark red on the reverse side. Conidia were obclavate or pyriform, olivaceous to dark brown, with 0 to 6 transverse septa and 0 to 4 longitudinal septa, 13.2 to 55.0 (27.9) × 6.3 to 12.5 (9.8) µm. Conidiophores were septate, hyaline to olivaceous brown, either branched or unbranched, geniculate at the tip, 2.5 to 5.9 μm wide and up to 70 μm long. These morphological and cultural characteristics were consistent with the descriptions of Alternaria alternata (Fr.) Keissl. isolated from Apple (Elfar et al. 2018). To confirm the pathogenicity on Italian ryegrass, healthy plants (8-week-old) of cultivar Splendor grown in five pots filled with potting soil were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Plants in another, five pots were sprayed with sterilized distilled water as controls. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 25°C. At 14 days post inoculation, symptoms typical of brown to dark brown leaf spots developed on the plants inoculated with conidial suspension, whereas no symptoms on the control plants. The pathogenicity tests were carried out three times. The same pathogen was consistently re-isolated from inoculated leaves and confirmed by morphological characterization as described above. To further identify this pathogen, isolate HMCH-9 (=CGMCC 3.19924) was selected as a representative for molecular characterization. Following Woudenberg et al. (2015), the internal transcribed spacer regions 1 and 2 and intervening 5.8S rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPD), translation elongation factor 1-alpha (TEF), RNA polymerase second largest subunit (RPB2), and Alternaria major allergen (Alt) genes were partially amplified and sequenced. Sequences were deposited in GenBank (accession nos. MH567106 for ITS, MH567107 for GPD, MH567109 for TEF, MH567110 for RPB2, and MH567108 for Alt). BLAST analysis of all these five segments showed >99.8% identity with those sequences of ex-type isolate CBS 916.96 of A. alternata (Woudenberg et al. 2015). To our knowledge, this is the first report of A. alternata causing leaf spot on Italian ryegrass in China. The accurate identification of this pathogen would be useful for the prevention and control of leaf spot on Italian ryegrass in the future.

First report of Alternaria alternata causing leaf blight on little millet (Panicum sumatrense) in India.

Little millet (LM) is a minor cereal crop grown in the Indian sub-continent. During October 2018, dark brown, circular to oval necrotic spots surrounded by concentric rings were observed on the upper leaf surface of the LM (cv. VS-13) grown in the fields of the University of Agricultural Sciences, Bengaluru, India (13.0784oN, 77.5793oE). As the disease progressed, infected leaves became blighted. Disease incidence up to 53% was recorded in 3 fields of 0.4-hectare area each. Thirty symptomatic leaves were collected to isolate the associated causal organism. The margins of diseased tissue were cut into 5 × 5-mm pieces, surface-sterilized in 75% ethanol for 45 seconds followed by 1% sodium hypochlorite for 1 min, finally rinsed in sterile distilled water five times and placed on PDA. After 7 days of incubation at 25°C, greyish fungal colonies appeared on PDA. Single-spore isolations were performed to obtain ten isolates. Pure cultures of the fungus initially produced light gray aerial mycelia that later turned to dark grey. All isolates formed obclavate to pyriform conidia measured 22.66-48.97μm long and 6.55-13.79µm wide with 1-3 longitudinal and 2-7 transverse septa with a short beak (2.55-13.26µm) (n=50). Based on the conidial morphology, the fungus was identified as Alternaria sp. Further, the taxonomic identity of all ten isolates was confirmed as A. alternata using species-specific primers (AAF2/AAR3, Konstantinova et al. 2002) in a PCR assay. Later, one of the isolate UASB1 was selected, and its internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh), major allergen Alt a 1 (Alt a 1), major endo-polygalacturonase (endoPG), OPA10-2, and KOG1058 genes were amplified in PCR (White et al. 1990; Berbee et al. 1999; Woudenberg et al. 2015), and the resultant products were sequenced and deposited in the NCBI GenBank (ITS, MN919390; gapdh, MT637185; Alt a 1, MT882339; endoPG, MT882340; OPA10-2, MT882341; KOG1058, MT882342). Blastn analysis of ITS, gapdh, Alt a 1, endoPG, OPA10-2, KOG1058 gene sequences showed 99.62% (with AF347031), 97.36% (with AY278808), 99.58% (with AY563301), 99.10% (with JQ811978), 99.05% (with KP124632) and 99.23% (with KP125233) respectively, identity with reference strain CBS916.96 of A. alternata, confirming UASB1 isolate to be A. alternata. For pathogenicity assay, conidial suspension of UASB1 isolate was spray inoculated to ten healthy LM (cv. VS-13) plants (45 days old) maintained under protected conditions. The spore suspension was sprayed until runoff on healthy leaves, and ten healthy plants sprayed with sterile water served as controls. Later, all inoculated and control plants were covered with transparent polyethylene bags and were maintained in a greenhouse at 28±2 ◦C and 90% RH. The pathogenicity test was repeated three times. After 8 days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms were observed on non-inoculated plants. Re-isolations were performed from inoculated plants, and the re-isolated pathogen was confirmed as A. alternata based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. There is an increasing acreage of LM crop in India, and this first report indicates the need for further studies on leaf blight management and the disease impacts on crop yields.

Enhanced solar water disinfection using ZnO supported photocatalysts

ABSTRACTNano-structured ZnO photocatalysts on cellulose and polyester supports were developed for enhancing solar water disinfection (SODIS). The photocatalysts were fabricated by a two-step hydrothermal method, in which ZnO nanoparticles were synthesized and deposited on a cellulose or polyester support as a seed layer, followed by the growth of one-dimensional ZnO nanorods on the seed layer in a liquid bath containing zinc nitrate and hexamethylenetetramine as sources of precursors. The morphologies and phase compositions of the synthesized ZnO nanorods from different growth conditions were investigated with field emission scanning electron microscope and X-ray diffraction (XRD), respectively. The crystallinity size of the ZnO nanorods was in the range of 17–30 nm and increased with the precursor concentration. The XRD patterns also revealed that higher growth solution concentrations led to higher intensity of XRD peaks, indicating higher crystallinity. Additionally, to test for SODIS enhancement, experiments using 200-mL transparent polyethylene bags as SODIS reactors, with ZnO photocatalysts inside, and water samples containing 106 CFU of Escherichia Coli were conducted in a laboratory UVA setup. The photocatalyst with a polyester support resulted in a 15% higher disinfection efficiency than that of the one with a cellulose support. Moreover, a field test of enhanced SODIS was conducted in actual sunlight, using specially designed SODIS reactors containing ZnO photocatalysts with a polyester support. Nearly total disinfection (97–98% efficiency) was achieved within the first 15 min of every test. The treated water was also tested for zinc contents, which could be released from the photocatalysts, by ICP-OES. The results were lower than 2 mg/L.

Germination and vigor of seeds of Magonia pubescens A. St.-Hil. submitted to storage and pre-imbibition in water

Abstract: Timbó (Magonia pubescens A. St.-Hil.) is a species with ornamental potential, which can be used in composition of gardens, squares and especially in streets landscaping. It is also suitable for planting in degraded areas of permanent preservation. This work aimed to verify the effect of seed size, storage and pre-imbibition time on seed germination and vigor. The test design was completely randomized, in a plot scheme subdivided in time. The main plots were seed size (large and small), packaging (0.5 mm-thick transparent polyethylene and kraft paper bag), and time of imbibition in water (0, 12 and 24 hours); and as subplots, it was considerate the storage times (0, 3, 6, 9 and 12 months). The tests were carried out in five replications of ten seeds, and the evaluated variables were: water content of the seeds, percentage of germination (% G), mean germination time (MGT), shoot height (cm), collar diameter (mm), number of leaves, and fresh and dry mass of the aerial part (g). The size of the seed did not influence the germination percentage, but large seeds formed more vigorous seedlings. The storage did not affect seeds germination, nor the vigor of the seedlings. Seed pre-imbibition did not influence the percentage of germination, but the mean germination time decreased, and more vigorous seedlings were formed. In order to obtain more vigorous Timbó seedlings, the best option is to use large seeds. They can be stored for up to 12 months without losing vigor and pre-soaked to accelerate the beginning of germination, thus forming a more homogeneous lot of seedlings.

Effects of increased zooplankton biomass on phytoplankton and cyanotoxins: A tropical mesocosm study

Zooplankton are important biocontrol agents for algal blooms in temperate lakes, while their potential in tropical and subtropical environments is not well understood. The aim of the present study was to evaluate the influence of increased zooplankton biomass on phytoplankton community and cyanotoxins (microcystins and saxitoxin) content of a tropical reservoir (Ipojuca reservoir, Brazil) using in situ mesocosms. Mesocosms consisted of 50L transparent polyethylene bags suspended in the reservoir for twelve days. Phytoplankton populations were exposed to treatments having 1 (control), 2, 3 and 4 times the biomass of zooplankton found in the reservoir at the beginning of the experiment. Filamentous cyanobacteria such as Planktothrix agardhii and Cylindrospermopsis raciborskii were not negatively influenced by increasing zooplankton biomass. In contrast, the treatments with 3 and 4 times zooplankton biomass negatively affected the cyanobacteria Aphanocapsa sp., Chroococcus sp., Dolichospermum sp., Merismopedia tenuissima, Microcystis aeruginosa and Pseudanabaena sp.; the diatom Cyclotella meneghiniana; and the cryptophyte Cryptomonas sp. Total microcystin concentration both increased and decreased at different times depending on zooplankton treatment, while saxitoxin level was not significantly different between the treatments and control. The results of the present study suggest that zooplankton biomass can be manipulated to control the excessive proliferation of non-filamentous bloom forming cyanobacteria (e.g. M. aeruginosa) and their associated cyanotoxins.

Photosynthetic CO2 uptake by microalgae for biogas upgrading and simultaneously biogas slurry decontamination by using of microalgae photobioreactor under various light wavelengths, light intensities, and photoperiods

Anaerobic digestion not only produces raw biogas which needs to be upgraded, but also nutrient-rich waste stream biogas slurry which needs decontamination. Therefore, this research focused on the effects of various light wavelengths, light intensities, and photoperiods on biogas upgrading and simultaneously biogas slurry decontamination by using of microalgae photobioreactor. The microalgae photobioreactor was a transparent polyethylene bag (80cm×60cm×11cm). The results demonstrated that biogas upgrading and simultaneously biogas slurry decontamination was successfully achieved by the use of the photosynthetic CO2 uptake by microalgae photobioreactor. The optimal light wavelength was the mixed LED red:blue=5:5; whereas the optimized lighting control strategy was: low light intensity (300μmolm−2s−1) with long photoperiod (16h light:8h dark) for the time course of 0–48h, moderate light intensity (600μmolm−2s−1) with middle photoperiod (14h light:10h dark) for the time course of 48–96h, and high light intensity (900μmolm−2s−1) with short photoperiod (12h light:12h dark) for the time course of 96–144h. Its biogas CO2 removal efficiency was 85.46±6.25%. Its removal efficiency of chemical oxygen demand, total nitrogen, and total phosphorus were 85.23±8.32%, 87.10±7.55%, and 92.40±3.05%, respectively.

STORAGE OF PITOMBEIRA SEEDS [Talisia esculenta (A. St. Hil) Radlk - SAPINDACEAE] IN DIFFERENT ENVIRONMENTS AND PACKAGINGS

ABSTRACT This paper aimed to evaluate the effect of packaging, environments, and storage periods on physiological quality of Talisia esculenta (A. St. Hil.) Radlk seeds. Seeds were conditioned in PET transparent bottle, Kraft paper bag, and transparent polyethylene bag; they were stored in chamber (18 °C; 50% RH), in freezer (-21 ± 2 °C; 95% RH), and under laboratory natural environment (28 ± 5 °C; 65% RH). They were evaluated at the 0, 25th, 50th, 75th, and 100th storage day for moisture content, thousand seed weight, emergence percentage, emergence rate index, length, and dry mass of shoot and root. T. esculenta seeds should be stored in chamber or laboratory natural environment, packed in polythene bag up to 25 days. The Kraft paper bag and the PET bottle cannot be used for storing T. esculenta seeds. Freezer is not recommended for storing T. esculenta seeds because it makes them unviable.

Influence of bunch covers on appearance and maturity of banana cv. Jahaji under high density planting system

External appearance and routine availability of fruits are the prime criteria for good marketability of bananas. In Assam, high density planting (HDP) is considered as a profitable practice in banana cultivation. However, the days taken to maturity and pest infestation are two of the most important factors hampering the production and market value of bananas. In addition to it, fall of temperature during the winter months hampers the crop cycle of banana to a greater extent in HDP. Considering the above sited problems, an investigation was carried out in the Experimental Farm, Department of Horticulture, Jorhat during 2013–14 in order to find out the best bunch cover material among white non-woven polypropylene bag, blue non-woven polypropylene bag, transparent polyethylene bag, leno bag, gunny bag, dry banana leaves and control (no cover) in banana cv. Jahaji (AAA) under the HDP to overcome delay in maturity and improvement of bunch appearance. It was observed that all bunch covers, irrespective of colour and material, significantly improved the fruit appearance. The highest numbers of scars and highest percentage of affected fingers were obtained in uncovered bunch (16 and 51.67%). Bunch covers protected the bunch from insect infestation and reduced the number of scars by 60% over control. However, the lowest number of scars and lowest percentage of affected fingers were obtained in T1 (6.56 and 7.67%). The experiment inferred that bunch covers also positively influenced the crop duration. White non-woven polypropylene bag recorded the shortest shooting-harvest interval (98.56 days) and days taken to harvest (422.33 days), while control recorded the longest shooting-harvest interval (120.44 days) and days taken to harvest (448.83 days). Variation in temperature within the covers was considered responsible for such change.