16-hour Photoperiod Research Articles

Confirmation report of Bacterial Leaf Streak disease of rice caused by Xanthomonas oryzae pv. oryzicola in Senegal.

Xanthomonas oryzae pv. X. oryzicola (Xoc), the causal agent of Bacterial Leaf Streak (BLS), is considered as one of the most important emerging pathogens of rice in Africa. This disease is estimated as responsible of 20 to 30% yield loss (Sileshi et Gebeyehu 2021) and has been characterized in several west African countries including Mali and Burkina Faso since 2003 and more recently in Ivory Coast (Wonni et al. 2014, Diallo et al. 2021). Presence of BLS symptoms in Senegal were reported by Trinh in 1980 but, to our knowledge, BLS occurrence has never been validated further and no strain of Xoc have ever been isolated from Senegalese rice fields. Xoc is transmitted by seeds which contribute to its spread through the rice trade (Sileshi et Gebeyehu 2021). To confirm Trinh's observations, we surveyed rice fields between 2014 and 2016 in eight different regions where rice is produced in Senegal. Typical disease symptoms characterized by yellow-brown to black translucent leaf streaks sometimes along with exudates, were detected in fields of several regions and collected. Leaf pieces were successively sanitized, rinsed in sterile water, and symptomatic fragments were ground using the Qiagen Tissue Lyser System (QIAGEN, Courtaboeuf, France). The leaf powder was diluted in 1.5 ml of sterile water and incubated for 30 minutes at room temperature. Ten μl of the suspension was streaked on semi-selective PSA medium and incubated at 28°C for 3 to 7 days. Characteristic round, convex, mucous, straw-yellow Xoc candidate colonies were purified from six individual leaf samples from three distinct sites in the northern Senegal River Valley. To confirm their identity, isolated strains were tested for pathogenicity and molecular characterization. All isolates were subjected to the multiplex PCR developed for the identification of X. oryzae pathovars (Lang et al., 2010) and revealed the same PCR profile (two amplicons of 324 and 691 base pairs) similar to that of the Xoc reference strain BLS256. Leaves of 5-week-old plants of O. sativa cv. Kitaake were infiltrated with a needleless syringe containing a bacterial suspension set at an optical density of 0.5. Upon seven days of incubation under greenhouse conditions (27 ± 1°C with a 12-hour photoperiod), all infiltrated spots (2 spots on 3 plants per isolate) developed water-soaked lesions similar to those caused by control strain BLS256, except when leaves were infiltrated with water. Symptomatic leaf tissues were ground and plated on PSA medium, resulting in colonies with typical Xanthomonas morphology that were diagnosed as Xoc by multiplex PCR typing, thus fulfilling Koch's postulate. At last, four of the isolates were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F and XgyrB1R (Young et al., 2008). Analysis of 780bp partial gyrB sequences of strains S18-3-4, S23-1-12, S52-1-4 and S52-1-10 highlighted 100% identity with the gyrB sequence of strain BLS256 (Acc. No. CP003057). To our knowledge, this is the first report of BLS in Senegal which is supported by molecular characterization methods. This study validates the presence of BLS in Senegal and will serve as a basis for future efforts of rice breeding for locally adapted resistance. More studies are needed to clarify the spatial distribution and prevalence of BLS in Senegal as rice cultivation is expanding rapidly in the country.

Germination Response of Three Florida Panhandle Coastal Dune Plants to Salinity

Campbell-Martínez, G.E.; Thetford, M., and Miller, D., 2022. Germination response of three florida panhandle coastal dune plants to salinity. Journal of Coastal Research, 38(3), 585–591. Coconut Creek (Florida), ISSN 0749-0208. Coastal dunes and their ecosystem services are threatened by human pressures warranting active restoration. Understanding dune plant germination responses to salinity is important when considering plant species and direct seeding locations for coastal dune restoration projects. The germination response of three Florida panhandle native coastal dune plant species to various salinity concentrations (0%, 0.25%, 0.50%, 0.75%, and 1% weight per volume) were tested. Species included were coastalplain honeycombhead (Balduina angustifolia (Pursh)B.L.Rob.), coastalsand frostweed (Crocanthemum arenicola (Chapm.)Barnhart), and squareflower (Paronychia erecta (Chapm.)Shinners). Seeds were germinated within germination boxes, placed in growth chambers under species-specific temperatures with a daily 12-hour photoperiod, and monitored weekly for 4 weeks. Germination responses to salinity were species-specific. Coastalplain honeycombhead had intermediate salt tolerance (highest germination at 0%–0.50% salinity), coastalsand frostweed was the least salt tolerant (highest germination at 0%–0.25% salinity), and squareflower was the most salt tolerant species in respect to germination (high germination across all salinities). Germination was delayed for all species at the highest salinity concentrations (0.75%–1%). However, for all three species, at least one-third of all seeds (>39%) at all salinity concentrations germinated by week 4. Results suggest germination for seeds of these species is possible in saline conditions documented within coastal areas before and after hurricane overwash. Future dune restoration studies for these species should test direct seeding of these plants under field conditions.

Фотопериодическая регуляция образования крылатых форм у свекловичной тли (Aphis fabae Scopoli, 1763)

The study of the process of migration in aphids has great practical importance. Thus, the aphids pass from one plant to another by these morphs and feed there and maintain their population by growing. The literature information on the research of the photoperiodic reactions in aphids is sufficient. However, the seasonal adaptation characteristics of sugar beet aphids have not been studied up to date in Azerbaijan. For this reason, the study of an impact of photoperiod on the emergence of winged ones in the population of black bean aphid is one of the important issues. During the study of reaction of individuals fed in the same routine against the photoperiod in the next generations, it has been defined that short photoperiod is more observed in the following generations. It seems that the information collected from the former generations is directly passed to the next generations. The results show that the continuous breeding of these species by means of parthenogenesis is only available in the long day (14, 16, 18, 24 hours) routine. However, the winged individuals prevail in the short photoperiods. The preference of winged individuals in 24 hours of photoperiod rather than others (14–18 hours) is probably related to the physiological changes occurs in them.

First Report of Brown Leaf Spot of Rice (Oryza sativa) Caused by Bipolaris sorokiniana in Pakistan.

Brown leaf spot of rice is one of the major seed-borne diseases and can diminish grain production up to 52% (Barnwal et al. 2013). In 2018, infected leaf samples showing the typical symptoms of brown spots were collected from the vicinity of the University of Agriculture, Faisalabad (31°26'10.3"N 73°03'35.1"E). The symptoms were brown-dark spots, with gray-light gray or brown centers surrounded by dark margins and with chlorotic halos and of oval or cylindrical shapes (5 to 9 mm in diameter). Disease incidence averaged 61% across the seven fields observed. Leaves were collected from the seven infected fields and symptomatic leaf tissues of 5 mm2 were excised from representative necrotic spots in each. These tissues were surface disinfected with 70% ethanol, rinsed with sterile distilled water (SDW), dried by blotting on paper, and placed on potato dextrose agar medium. For pathogen growth, the plates were placed at 25oC (±2oC) with a 12-hour photoperiod for 5 days. Five samples from each of the infected fields were taken for pathogen isolation and among them ten isolates were sub-cultured and purified by using the single spore method. The resulting fungal colonies were fluffy and ranged in color from grayish black/black to light brown. Fifteen conidia were measured that are olivaceous-brown to dark brown in color, elliptical to oblong with narrow (tapered) ends, with 3-10 septa and 35.6-65.4 µm in length x 13.1-25.7 µm in width. Conidiophores were yellowish-brown, geniculate, and solitary (Pratt 2003). For molecular studies, rDNA of the internal transcribed spacer (ITS) region, translation elongation factor (tef), RNA polymerase II second largest subunit (rpb2) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified by using the primers ITS1F/ITS4R (White et al. 1990), EF1-983F/EF1-2218R (Rehner and Buckley 2005), 5F2/7CR (O'Donnell et al. 2007), and GPD1/GPD2 (Berbee et al. 1999) respectively. The sequence of all the amplified gene regions of one SUL-1 isolate was deposited into GenBank with accession numbers MN314844 (ITS), MN326866 (tef), MN990457 (rpb2) and MN990456 (gpd). BLASTn queries of the obtained sequences (ITS, tef, rpb2 and gpd) showed 99-100% homology with the corresponding nucleotide sequences of B. sorokiniana (GenBank accession nos. GU480767, MF490855, LT715652 and MK558818 respectively). To fulfill the Koch's postulates, twenty rice plants (cv. Basmati-385) were sprayed at 2 to 3 leaf stages by using the two representative isolates with a spore suspension of 105 spores/ml. SDW was sprayed on ten control plants. The plants were covered with polyethylene bags to keep the moisture contents and incubated at 25oC (±2oC) for 7 days. After a week, same symptoms as those described above were observed. In the repeated experiment, B. sorokiniana was re-isolated from the infected rice leaves and confirmed morphologically; fulfill the Koch's postulates. With grave worry, the other species of the genus Bipolaris (B. oryzae, and B. victoriae) have also been found to the cause brown leaf spot of rice (Motlagh and Kaviani 2008). To our knowledge, this is the first report of Bipolaris sorokiniana causing brown leaf spot of rice in Pakistan. Because rice is highly consumable grain in Pakistan, so the rapid spread of this disease in the rice farming areas is of a serious concern.

First Report of Phyllosticta capitalensis Causing Black Freckle Disease on Rubus chingii in China.

Rubus chingii is used as an important traditional Chinese medicine, and belongs to the family Rosaceae. The fruit has multiple pharmacological activities, including antioxidant, anti-inflammatory, and improving cognitive impairment (Na Han et al. 2012). In June 2019, a new fungal infection was observed on the leaves of R. chingii in Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province, China, forming small lesions with reddish-brown edges along leaf veins. Over 500 plants were surveyed, and nearly 20% of the plants were symptomatic. The diseased plants grew poorly and appeared stunted, and severely affected plants died. Five symptomatic leaves were randomly collected from the field and washed with tap water and distilled water successively. The edges of infected leaf tissue were cut into small pieces (4 to 5 mm2), surface sterilized with 70% ethanol for 30 s and 0.1% HgCl2 for 1 minute, and then rinsed three times in sterile distilled water (Chen et al. 2016). The same fungus was isolated from 41 pieces. The hyphae of a representative isolate were gray, the colony surface was granular, the edges were uneven and white, and the culture turned black over time with black spherical conidia. Conidia were nearly elliptical, unicellular, and each with a hyaline, unstable apical appendage, 3 to 10 µm long. The size of conidia was 10 to 18 μm in length and 4 to 8 μm in width. These morphological characteristics are consistent with those described for the fungus Phyllosticta capitalensis. (Wikee et al. 2013). For an accurate identification, genomic DNA of a representative isolate of the pathogen was extracted to amplify the internal transcribed spacer (ITS) region, the transcription elongation factor (tefa-1), and actin (ACT) genes with the ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Cheng, L. L. ,et al. 2019), respectively. The ITS, tefa-1 and actin gene sequences were deposited in GenBank and assigned accession numbers MW308365, MW714380 and MW714381, respectively. BLAST search analysis of GenBank (NCBI) showed that the sequences had 100% similarity with those of Phyllosticta capitalensis (GenBank accession no. ITS, MN548091; tefa-1, MN958711; and ACT, MN565575). The pathogenicity of Phyllosticta capitalensis was verified using six healthy detached leaves from healthy R. chingii plants around 40 cm tall. A total of nine plants were used, and three leaves from each plant were artificially inoculated. Each wound was inoculated with conidial suspension (106 mL-1), while the control leaves were coated by sterile water. All the treated plants were covered with plastic bags for 2 days, incubated at 28ºC and 85% relative humidity, with a 12-hour photoperiod. After 15 days following inoculation, the injured leaves showed similar symptoms to the above-mentioned lesions, while the control and uninjured leaves were still healthy. P. capitalensis were reisolated from inoculated leaves, fulfilling Koch's postulates. P. capitalensis is an endophyte, widely distributed in various host plants in China. (Lu, J. M, et al. 2016). To the best of our known, this is the first report of black freckle disease caused by P. capitalensis on Rubus chingii in China. P. capitalensis is a destructive plant pathogen with an unusually broad host range and our findings will be useful for its management and for further research. The author(s) declare no conflict of interest.

Features of long-term preservation of Azhurnaya and Bergamotnaya mint cultivars in an in vitro collection

Mint represents a valuable medicinal and essential oil plant widely used in pharmacology, perfumery, cosmetics, and food industry. In order to maintain collections of cultivars and valuable samples, as well as to carry out breeding, the application of biotechnology appears to be promising for in vitro preservation of plant objects under the conditions of slow growth at low positive temperatures. The present study is focused on the morphometric parameters and genetic stability of the explants of two mint cultivars during their in vitro preservation at 4–6 ºС for a year without illumination. Meristems with two leaf primordia were cultivated on a nutrient medium in a culture room at 26 ºС and illumination of 2–3 klx with a 16-hour photoperiod. Microshoots developing from meristems were divided into single-node stem segments to be used as explants for deposition. After a year of deposition at 4–6 ºС without illumination, the number of viable explants in the Azhurnaya and Bergamotnaya cultivars was established to be 56.5 and 85.7%, respectively. The explants under study were characterized by the development of up to 2.5 shoots up to 13.2 mm long and rhizogenesis with a frequency of up to 52.3%. Following the deposition, microshoots were cut and transferred to a culture room to resume growth. After two subcultivations, viability (100%), morphometric parameters of microshoots, and multiplication index (up to 5.5–7.3) were fully restored. The ISSR-analysis performed according to four studied DNA markers revealed the microshoots of these cultivars to be completely identical to the original plants after in vitro preservation. The conducted studies proved the effectiveness of the conditions used for in vitro preservation of mint explants and confirmed the explant genetic stability after a year of deposition at 4–6 ºС.

GERMINATION OF CACTUS SEEDS UNDER SALINE STRESS

ABSTRACT Cactus seeds in seasonal dry tropical forests are subject to several stressors, such as salt stress which limits imbibition and therefore germination. Thus, this study aimed to compare germination performance of Cereus jamacaru subsp. jamacaru and Pilosocereus pachycladus subsp. pernambucoensis seeds under salinity conditions. To this end, NaCl and KCl solutions were used in the following osmotic potentials: 0.0 (control),-0.2,-0.4,-0.6,-0.8,-1.0, and-1.2 MPa. Seeds were placed to germinate at 25 °C and 12-hour photoperiod. The number of germinated seeds was counted daily for 21 days after root protrusion. The variables analyzed were: water content, germination, normal seedlings, germination speed index, and average germination time. The experimental design was completely randomized, following a 2 × 7 factorial scheme (species × osmotic potential) for each saline source. Water restriction and ionic effect caused by salts favored seed germination and vigor in both species at-0.2 and-0.4 MPa. However, from-0.8 MPa onwards, germination decreased significantly for both salts. Seeds of C. jamacaru subsp. jamacaru and P. pachycladus subsp. pernambucoensis were tolerant to salt stress since they germinated up to-0.8 MPa (NaCl) and-1.2 MPa (KCl). The latter occurred only for P. pachycladus subsp. pernambucoensis.

First Report of Alternaria alternata Causing Leaf Spot on Chili (Capsicum annuum) in Pakistan.

Chili (Capsicum annuum L.) is an important vegetable crop in Pakistan. During summer of 2019, chili leaf spot symptoms were observed on 3-month-old plants in the fields, with 30 to 40% of disease incidence, in District Faisalabad, Punjab, Pakistan. Diseased leaves were characterized by numerous tiny round spots (0.5 to 2.0 mm in diameter, average 1 mm) that were white to grey with a sunken center, surrounded with dark brown edge and chlorotic halo. The lesions gradually enlarged and coalesced into large, nearly circular, or irregularly shaped lesions that could be as long as 3 cm. Small pieces of symptomatic leaf tissues were surface sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin (100 ppm). After 5 days at 25°C with a 12-hour photoperiod, same fungal colonies developed. The colonies initially appeared white and then turned olive-green. The conidiophores were brown septate and generally branched. Conidia borne singly or in short chains were multicellular, obclavate to obpyriform, and 16.2 to 38.5 µm (average 27.35 ± 2.1 µm) in length and 8 to 16.5 µm (average 12.25 ± 1.6 µm) in width, with zero to three longitudinal and two to five transverse septa (n=35). The fungus was identified as Alternaria sp. (Fr.) Keisel based on its morphological characteristics (Simmons et al. 2007). For molecular identification, genomic DNA of two representative isolates (SSUAF1 and SSUAF2) was extracted using DNAzol reagent and PCR amplification of the internal transcribed spacer (ITS)-rDNA region, Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) gene and RNA polymerase II second largest subunit (rpb2) were performed with primers ITS1/ITS4 (White et al. 1990), gpd1 and gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu, et al. 1999), respectively. The obtained sequences were deposited in GenBank with acc. nos. MT249008.1 and MT249009.1 for ITS-rDNA; MT318220.1 and MT318221.1 for the GAPDH; and MT318236.1, and MT318237.1 for RPB2 gene. A BLAST search in GenBank showed 100% identity with A. alternata for both ITS region (MT279999.1), GAPDH gene (MK637438.1) and RBP2 gene (MK605900.1). To confirm pathogenicity, 2-month-old healthy potted C. annuum plants were inoculated using an atomizer in a greenhouse. A total of 12 plants at the true leaf stage in each experiment were sprayed with a conidial suspension (106 conidia/ml) of both isolates amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant). Four plants were inoculated with each of the two isolates, whereas four control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse. After 10 days of inoculation, each isolate induced leaf lesions that were similar to typical lesions observed in the field. The experiment was conducted twice with similar results. The fungus was readily reisolated from symptomatic tissues whereas the control plants remained symptomless. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling the Koch's postulates. Previously, A. alternata has been reported in Italy and India (Devappa et al. 2016; Garibaldi et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot of C. annuum in Pakistan. This report will help the identification of leaf spot of chili and the development of management strategies for control of this disease in Pakistan.

Biology and Life-Table Parameters of Fall Armyworm, Spodoptera frugiperda on Three Maize Cultivars Grown in Thailand

Abstract Fall armyworm, Spodoptera frugiperda has become a new invasive species in Thailand that mainly affects maize. There has been limited research on the baseline biological aspects of this insect under controlled conditions. Our objective was to detail the biological parameters of S. frugiperda reared on maize (Zea mays L.) cultivars, field maize, sweet maize and waxy maize under controlled conditions at 30 ± 2°C, 55 ± 5% RH and a 12-hour photoperiod. Results suggest that larvae develop through six instars on all the maize cultivars. Significant effects (P < 0.05) of the host plant were found in the duration of larval stage 10.83 ± 0.14, 11.15 ± 0.15 and 11.28 ± 0.05 days when fed with sweet maize, waxy maize and field maize, respectively. More than 70.5% of them transformed into the pupal stage. The life cycle duration lasted 28.11 ± 0.40 days (field maize), 27.16 ± 0.37 days (sweet maize) and 28.41 ± 0.34 days (waxy maize). Significant differences among host plants were not observed for the different development durations. S. frugiperda exhibited a type I survivorship curve. The highest values of net reproductive rate, R0 (220.41 ± 5.88), innate capacity of increase, rc (0.23 ± 0.001) and finite rate of increase, λ (1.25 ± 0.002) obtained on sweet maize, were not statistically different from other cultivars. The mean generation time (Tc) was significantly different among the 3 maize cultivars, ranging from 26.36 ± 0.43 days on waxy maize to 23.80 ± 0.24 days on sweet maize. Information presented here should greatly expand the understanding of S. frugiperda biology; this understanding can be used to improve the efficiency of management techniques for this critical crop pest. Keywords: Invasive species, Maize pest, Biological cycle, Reproductive rate, Population growth

First Report of Aphanomyces euteiches Race 1 and Race 2 Causing Aphanomyces Root Rot on Alfalfa (Medicago sativa) in South Dakota.

Aphanomyces euteiches causes Aphanomyces root rot (ARR) in alfalfa (Medicago sativa), along with root rot on many other legumes, including pea, clover, and lentil (Malvick et al., 2009). In 2020, South Dakota (SD) planted the most acres of alfalfa in the United States, which demonstrates the importance of alfalfa to the state. Several SD growers reported alfalfa establishment problems likely to be associated with ARR. Soil samples were collected from 16 fields under commercial alfalfa production in Lake County, SD in June 2020. Composite soil samples based on 24 subsamples were collected in a W-shaped pattern at a depth of 15 cm. Collected soil was sieved, and 80 cm3 was placed in plastic pots (6 cm x 6 cm). Each pot was planted with 25 seeds, covered with an additional 15 cm3 soil, and placed in a growth chamber with a 16-hour photoperiod at temperatures of 24 and 19 ℃ (day and night). Alfalfa seedlings, including Saranac (susceptible to races R1 and R2), WAPH-1 (resistant only to R1), WAPH-5 (resistant to both R1 and R2), and Mustang 625 (resistant to both R1 and R2 and coated with mefenoxam) grew in collected soil for 7 days, followed by 4 days under flooded conditions. Trays were drained, and at 21 days after planting (DAP), roots were removed from soil, washed in distilled water, and rated to measure severity of disease symptoms (Samac et al., 2015). The average severity index (ASI) used a 1-5 disease severity scale, 5 being a dead plant and 1 being no symptoms present (http://www.naaic.org/stdtests/Aphano.html). Race was based on ASI where R1 included an ASI of ≥3 for Saranac and <3 for WAPH-1, and R2 included an ASI of >3.0 for Saranac and WAPH-1 and <3.0 for WAPH-5 (Malvick and Grau, 2001). Race-typing experiments were repeated twice with six replicate pots per alfalfa cultivar per experiment and determined the presence of both R1 and R2 in Lake County, SD. ASI values for Mustang 625 and WAPH-5 were similar across all fields evaluated, which indicates limited confounding effects of other root rotting pathogens. DNA was extracted from three symptomatic roots from each field and was PCR amplified using A. euteiches specific primers (Vandemark et al., 2002). A PCR product was observed in all 16 fields evaluated, and the absence of a product was observed when DNA was extracted from alfalfa roots grown in vermiculite. Following race-typing, infected alfalfa roots were surfaced sterilized and placed on Aphanomyces selective media consisting of mefenoxam and benomyl in cornmeal agar (CMA) (Pfender et al., 1984). Isolates were identified as A. euteiches based on hyphal morphology (Malvick and Grau, 2001). Alfalfa seedlings (Saranac) were grown in vermiculite under growth conditions used for the race-typing assay and inoculated 6 DAP with two isolates of A. euteiches. Inoculation was completed using half plates of one week old A. euteiches mycelium on CMA blended with one liter of water (Samac et al., 2015). At 35 DAP, control alfalfa seedlings inoculated with blended CMA and water remained asymptomatic, and alfalfa infected with A. euteiches displayed symptoms including honey-brown colored lesions. For confirmation of Koch's postulates, DNA from three re-infected seedlings was again PCR amplified using A. euteiches specific primers and confirmed our previous work. This is the first report of either R1 or R2 of A. euteiches causing ARR on alfalfa in SD. To avoid future yield loss, SD growers should consider planting available alfalfa cultivars that have resistance to both races of A. euteiches.

INFLUENCE OF BISPHENOL A AND PROBIOTIC-CONTAINING FEED CARASSIUS GIBELIO BLOCH INDICATES SEPARATELY

One of the most common pollutants of water bodies is bisphenol A (BPA), a plastic monomer used to synthesize polycarbonate plastics, epoxy resins and thermal paper (Barboza L. et. al., 2020). BPA enters freshwater and marine ecosystems as a result of leaching from BPA-based composites, as well as with water discharges from manufacturing plants, wastewater treatment plants and landfills. BPA is characterized by a low accumulation potential in freshwater environments, a short half-life under aerobic conditions, but due to the continuity of exposure, it is a serious problem (Wu N. C., Seebacher F., 2020). The ways BPA enters the fish body are diverse: through the digestive tract, gills, skin. Numerous studies have shown the adverse effects of BPA on the behavioral and morpho-physiological parameters of fish, including swimming patterns, coordination of movements, appetite, and dysfunction of many systems (endocrine, reproductive, nervous). It is possible to prevent the negative effects of hunting through the prophylactic use of agents that enhance the overall reactivity of the body. In this sense, our attention was drawn to probiotics - microorganisms that have a stimu-lating effect on the development of indigenous microflora, have a high antagonistic, synthetic, immunomodulatory, regulatory potential. We have studied the effect of bisphenol A and probiotic microorganisms Lactobacillus casei, introduced in the feed, on certain indicators of Carassius gibelio Bloch (general behavioral reactions, certain mor-pho-physiological parameters, qualitative and quantitative characteristics of erythrocytes and leukocytes). The study was carried out after 15-day acclimatization of fish in aquariums at a water temperature of 14°C, appropriate aera-tion regime and 16-hour photoperiod. It was found that 96-hour exposure to bisphenol A at a concentration of 1.5 mg/l causes darkening of the skin, in-creased mucus secretion, uncharacteristic motor activity, the appearance of unusual morphotypes of red blood cells, a slight increase in the number of leukocytes, and a decrease in the phagocytic activity of Carassius gibelio Bloch. Preventive introduction of probiotic cultures in the feed contributed to the correction of behavioral reactions and individual hematological parameters

Organogênese in vitro como método eficiente para a propagação de Dalbergia nigra

Abstract The objective of this work was to establish an efficient protocol for the in vitro organogenesis of Dalbergia nigra. For this purpose, 30-day-old seedlings were sectioned at their cotyledonary nodes and nodal segments. These materials were cultivated in a medium with different combinations of the 6-benzylaminopurine and thidiazuron cytokinins. After 60 days in a growth chamber set at 27°C and a 16-hour photoperiod, growth characteristics were analyzed. Explants from the cotyledonary nodes show a greater morphogenetic potential, regardless of the addition of cytokinins. Cotyledonary nodes in the medium supplemented with 2.22 µmol L-1 BAP show the best combination for the in vitro propagation of D. nigra.

First Report of Fusarium graminearum Causing Flower Blight On Hemp (Cannabis sativa) in Kentucky.

In October of 2020, a grower in Boyle County, KY, reported mold and blight symptoms on flowers of field-grown hemp. Plants were approaching harvest, and the mold was affecting 100% of the cultivar 'White CBG' being grown for cannabinoid (CBD) extraction. Mycelium colonized the flower heads and any seeds within bracts. Affected flower bracts were necrotic, and mycelium and necrosis in the most severe cases also encompassed adjacent (sugar) leaves. Necrotic symptomatic tissue was collected, disinfested in 10% bleach for one minute, and cultured on acidified potato dextrose agar (APDA). Each isolate was single-spored, transferred to PDA, stored in 15% glycerol at -80°C and maintained at room temperature under blacklight blue and fluorescent bulbs on a 12-hour light-dark cycle. Colonies produced white-pink mycelia with a dark red pigment on the undersides. Conidia collected after 7-9 days were falcate and septate (5 to 6). No microconidia were produced. Macroconidia measured 35.4-49.7 µm x 3.4-5.8 µm (n=50). The strains produced blue-black fertile perithecia on carrot agar when induced according to the method of (Bowden and Leslie, 1999). To confirm pathogenicity, flowers of hemp cultivars 'Lifter', 'Trump Towers', 'Wife' and 'White CBG' were inoculated in the greenhouse with a representative fungal strain (20Hemp010). Plants were inoculated at two different stages: when the styles were still green or after they had become senescent. Macroconidia were collected from 7- to 9-day-old cultures grown under a 12-hour light-dark cycle. Plants were spray-inoculated with a 5 x 105 per ml conidial suspension in 0.05% Tween 20 until runoff. Flower heads were individually covered with clear plastic bags and incubated for 72 h at 95-100% humidity under greenhouse benches to avoid direct light. Bags were removed after 72 h and returned to the bench. Greenhouse conditions were 23-25°C with a 14-hour photoperiod and 50% RH. Symptoms developed 7 dai in 1% of the flowers inoculated when styles were green, and 36% of the flowers that had senescent styles. Symptoms were similar to those initially noticed in Boyle County, including necrotic flower bracts and sugar leaves, and visible fungal growth. Symptoms were more severe on plants inoculated when styles were necrotic. Recovered fungi were morphologically similar to 20Hemp010. Genomic DNA was extracted from the mycelium with the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit. A fragment of the translation elongation factor 1-alpha 1 gene was amplified with primers EF1 and EF2 as described by (O'Donnell et al. 1998). Amplicons were sequenced and the consensus (MZ407909) was compared with the NCBI GenBank Refseq database by BLASTn. The top hit was Fusarium graminearum with 100% identity (JF270185.1). Pairwise alignments via MycoBank Fusarium MLST and Fusarium-ID also revealed a top hit of F. graminearum with 100% identity (AY452957.1). Conidial and colony morphology were also consistent with F. graminearum (Leslie and Summerell, 2006), thus we conclude that this species was the causal agent of the flower blight and mold. The same disease was subsequently confirmed on hemp in Breathitt and Franklin Counties in KY in 2020. This is the first report of this disease in KY, although F. graminearum has been reported previously causing a similar flower blight on hemp in NY and NC (Bergstrom et al., 2019, Thiessen et al. 2020). Fusarium graminearum is common in KY as a cause of Fusarium head blight on wheat and Gibberella ear rot on corn. In cereals, fungal infection is facilitated by the production of the mycotoxin deoxynivalenol (DON), which is harmful to humans and livestock (Desjardins and Hohn, 1997). As hemp production in Kentucky continues to rise for production of CBD products and edible grains, accumulation and concentration of DON in these products could become a concern.

First report of banana freckle disease caused by Phyllosticta cavendishii in Mauritius

Banana (Musa sp.) is among the most important fruit crops grown in Mauritius with significant socioeconomic and cultural considerations. The annual production of banana approximates to around 8000 tonnes (Statistics Mauritius, 2018). Dwarf Cavendish is the main variety grown commercially in Mauritius. During the hot and humid month of February 2014, symptoms consisting of dark black spots were observed on unripe banana of Dwarf Cavendish from the region of Nouvelle Découverte (super humid zone). A disease incidence of more than 50% with a moderate to severe level of severity across the field was reported by the extension services of the Food and Agricultural Research and Extension Institute. On the fruits, the initial symptom was tiny reddish-brown spots, bordered by dark green water-soaked margins (Figure 1). As the spots enlarged and coalesced, the surface became rough and turned black. These spots had a rough texture like sand-paper, similar to the description described by Wong et al. (2012). Comparable symptoms were observed on the leaves (Figure 2). Results from a rapid island-wide survey showed that only the Cavendish-type banana varieties displayed symptoms of infection. Out of 98 banana fields surveyed (50.9 ha), 65% were affected. Morphological identification of the suspected fungal pathogen was done using a compound light microscope. A sterile needle was used to tease pycnidia on leaves in a drop of sterile water to release conidia, before being mounted in clear lactic acid for microscopic examination. Conidia (Figure 3) formed singly at the apex of the conidiogenous cell were hyaline, aseptate, oblong to ellipsoidal, coarsely guttulate and measured 7.1-15.6 × 4.4-8.3 μm. The apical mucilaginous appendage arising from the conidium was straight to curved and was 5.1-12.9 μm long. Spermagonia were 70–125 μm in diameter and 50–85 μm high. Dumbbell-shaped spermatia (Figure 4) were hyaline, aseptate, biguttulate and measured 7.5-9.5 × 1.3-1.8 μm. These morphological characters are consistent with the species description of Phyllosticta cavendishii (Wong et al., 2012). Lesions from 11 samples of leaf and fruit with fruiting bodies were ground in liquid nitrogen followed by genomic DNA extraction. Extraction of DNA was done on c. 50 mg fresh plant tissue using the Biosprint 15 DNA Plant Kit (Qiagen, Germany). Tissues were first homogenized using a Tissuelyser (Qiagen, Germany) in a 2 ml microcentrifuge tube containing a 3 mm metal bead for two 30 second bursts at 30Hz. Two microliters of the eluted DNA was used as template in an ITS PCR amplification using AmpliTaq Gold 360 Mastermix (Applied Biosystems, USA) and 0.5 μM each of primers GMF1 and GMR2 (Wong et al., 2012). Thermal cycling conditions were 95°C for 10 min followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 1 min. Final extension of PCR products was done at 72°C for 10 min. Amplicons (550bp) were directly sequenced using the same primer pair. Resulting sequences were analysed using BlastN as well as by manual comparison to known sequences of Phyllosticta isolates which cause freckle disease of banana. The causal agent from all 11 samples tested was found to be Phyllosticta cavendishii. The ITS sequences were deposited in GenBank (Accession Nos. MW520341-MW520351) and at the Queensland Plant Pathology Herbarium (BRIP 72048 to 72058). Pathogenicity was tested using twenty mature green healthy fruits of banana variety Dwarf Cavendish. Fruits were surface sterilised using 0.2% sodium hypochlorite for 2 mins, rinsed with sterilised distilled water and allowed to dry before inoculation. Ten fruits were artificially inoculated on the outer surface, clearly marked, with a conidial suspension of 106 conidia/ml, prepared from pycnidia extracted from field infected tissue. Control fruits were treated with sterile distilled water. All the fruits were covered with plastic bags for 48 hours and incubated at room temperature, 80% relative humidity and with a 12-hour photoperiod. After 7 days, typical minute black spots identical to those observed in the field, appeared on inoculated fruits, whereas controls remained healthy. The morphological characteristics of the fungus were consistent with those previously observed, hence fulfilling Koch's postulates. To our knowledge, this is the first report of Phyllosticta cavendishii causing banana freckle disease in Mauritius. The authors acknowledge the Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation for the molecular identification of Phyllosticta cavendishii and also Dr Yu Pei Tan from the Brisbane Plant Pathology Herbarium for accessioning isolates and submission into GenBank.

Inter - and intraspecific relationships between Macrosiphum rosae (Hemiptera: Aphididae) and Chrysoperla externa (Neuroptera: Chrysopidae)

The predator Chrysoperla externa (Hagen, 1861) (Neuroptera: Chrysopidae) is a polyphagous species that has, among its wide range of acceptable prey, the aphid Macrosiphum rosae (Linnaeus, 1758) (Hemiptera: Aphididae), an important pest of rose plants. We assessed the consumption of nymphs and adults of M. rosae by C. externa larvae, the predatory efficiency, and larval survival under different predator:prey ratios, densities of aphids, and ages of aphids. The bioassays were performed at 25 ± 1°C and 70 ± 10% RH under a 12-hour photoperiod, with aphids taken from roses of the cultivar Avalanche. Consumption was assessed by making young aphids (2nd and 3rd instars) and more developed aphids (4th instar and adult) available throughout the larval stage of the predator. The predatory efficiency and survival were studied for second-instar larvae offered M. rosae nymphs at densities of 20, 40, 80, 120, and 160 using predator:prey ratios of 1:5, 1:10, and 1:20. The assessments were performed 24, 48, and 72 hours after putting the species together. All of the C. externa instars consumed younger than older aphids. Larvae in the third instar consumed 80% of the young aphids and 77% of the older aphids. The higher the predator:prey ratio and larval density, the higher the predatory efficiency and the lower the larval survival. The ratios 1:5 and 1:10 led to the elimination of younger and older aphid populations 72 hours into the interaction between species.

First report of Pucciniastrum minimum (syn. Thekopsora minima) causing leaf rust on Vaccinium corymbosum (blueberry) in the United Kingdom and pathogenicity on Vaccinium myrtillus (bilberry)

In September 2021, locally grown northern highbush blueberry plants (Vaccinium corymbosum cv. ‘Liberty’) from a nursery in Perthshire, Scotland were discovered to have rust-like disease symptoms (Figures 1, 2). Two of five of the plants appeared to be infected, with less than ten leaves on each plant showing symptoms. Each plant showed characteristic yellow-orange hypophyllous uredinia with urediniospores on the lower leaf surface and circular lesions of necrosis on the upper leaf surface. While defoliation was observed, it is unknown if this was associated with the rust infection. Morphological observations determined that urediniospores collected from fresh pustules were subglose, oblong or ellipsoid measuring 9.1–11.9 μm in width, and 11.8–19.2 μm in length (n = 40; Figure 3). Urediniospores were entirely echinulate, unlike the urediniospores of Naohidemyces fisanensis. Since Pucciniastrum and Naohidemyces have only subtle differences in the uredinial stage (Sato et al., 1993), urediniospores were collected for DNA extraction and sequence-based identification. The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was then amplified and sequenced with ITS5 and ITS4 primers (White et al., 1990). A BLAST analysis revealed 99.8% identity to a Thekopsora minima (synonym of P. minimum) isolate from Germany (GenBank Accession No. MN880392; Figure 4). The 671 bp long sequence was deposited in GenBank under Accession No. OK625817. To fulfil Koch's postulates, detached leaves from healthy blueberry cv. ‘Bluecrop’ and wild bilberry (Vaccinium myrtillus), a native Vaccinium sp. in the UK, were surface sterilised with 70% ethanol for two minutes, followed by rinsing with sterile distilled water. Fresh urediniospores from the original symptomatic leaves were then rubbed against the underside of the sterilised leaves. Uninoculated controls comprised sterilised healthy leaves lightly rubbed with leaves without spores. Leaves were placed in a tray lined with a damp paper towel, sealed in a polyethene bag to maintain humidity and incubated at 22°C (12-hour photoperiod, LED lights). After 20 days, yellow-orange uredinia formed in small clusters on inoculated blueberry and bilberry leaves. The clusters were much smaller on bilberry than on blueberry. Microscopy identified the presence of urediniospores on both hosts (Figure 5). No symptoms were observed on the control leaves. Bilberry is a widespread native species in the UK with particular ecological importance on heathland. The ability of P. minimum to cause disease in V. myrtillus under laboratory conditions is particularly noteworthy due to the current significant threat of Phytophthora kernoviae and Phytophthora ramorum on bilberry populations. The pathogen has been previously reported on highbush blueberry in China, Germany, New Zealand, South Africa, South America and United States (e.g. Padamsee & McKenzie, 2019; Pazdiora et al., 2019; Wichura et al., 2020). To our knowledge, this is the first report of P. minimum in the UK and the first time V. myrtillus has been identified as a host.

Effects of Photoperiod Alterations on Stress Response in Daphnia magna

In the modern world people are often impacted by factors that control when they can sleep and for how long, which results in an altered photoperiod. Alterations in photoperiod can occur from a variety of factors, ranging from working night shifts to travelling and changing time zones, and therefore influence the daily lives of many people. The purpose of these experiments was to determine if alterations in photoperiod affect the stress response in Daphnia magna. We hypothesized that if Daphnia magna are exposed to alterations in photoperiod, then there will be an increased stress response. We kept two distinct Daphnia populations and exposed the experimental group to a shorter photoperiod of 12 hours instead of the traditional 24-hour photoperiod. During the testing period, we tracked possible stress responses, including mean heart rate, brood size and male-to-female ratio. There were statistically significant differences between the control and experimental groups for both heart rate and brood size. Specifically, the experimental group exhibited an increased heart rate and brood size relative to the control group. Within the experimental group, there was statistically significant variation in the brood size over time, with the peak brood size occurring on day three or four in the seven-day trial. Male-to-female ratio did not have a statistically significant response to the altered photoperiod. The results found support the hypothesis of increased stress when photoperiod is altered, which could also indicate that altered photoperiod may have an impact on people that constantly experience changes in their photoperiod.

Germination of coastalplain honeycombhead (Balduina angustifolia) in response to photoperiod, temperature and gibberellic acid

Coastalplain honeycombhead (Balduina angustifolia) is underutilised in ecosystem restoration and pollinator conservation projects. Seeds collected from different populations throughout Florida, USA during 2007 and 2016-2018 were used in experiments that evaluated the effects of a 0 or 12-hour photoperiod, seasonal and standard temperatures, 0-5,000 ppm gibberellic acid (GA) or source population (northwest, central and south Florida) on seed germination. For central Florida seeds, germination was high (62-74%) in all seasonal temperatures except for summer (4%) and was not affected by photoperiod. Germination differed for two northwest populations and was higher in warm (35/25 and 30/20°C) temperatures compared with cool (25/15 and 20/10°C) temperatures (63-72% vs. 9-36%). In a one-year move-along experiment, germination was similar at all seasonal and constant temperatures for seeds from central, south-central and south Florida, while northwest Florida seeds had reduced germination under winter temperatures. Germination of northwest Florida seeds after exposure to GA increased for two of four and three of four populations for one year and five-day old seeds, respectively. There were different temperature requirements on a regional scale and different dormancy levels at a local scale for coastalplain honeycombhead.

BREAKING SEED DORMANCY IN Sesbania punicea (Cav.) Benth

The objective of this study was to evaluate methods of breaking dormancy in Sesbania punicea seeds for seedling production. The treatments used were control, manual scarification for 40 s, manual scarification for 60 s, thermal scarification and chemical scarification. After the treatments were carried out, the seeds were placed to germinate at 25 ºC and a 16-hour photoperiod for 14 days. The germination percentage, average germination time, germination speed index, percentage of normal seedling formation, shoot length, root system length and percentage of hard seeds were evaluated. A completely randomized design with four replications of 25 seeds was used. The data were submitted to ANOVA and the means compared by Tukey 5%. There was a difference for all the analyzes evaluated, except for the length of the aerial part. The treatment with manual scarification 60 s had superior results, being indicated for overcoming dormancy of seeds of S. punicea.

Optimization of microclonal reproduction of potato wart cultivar-differentiators

Goal. To study the influence of ammonium salts of dihydropyrimidine to the formation of microtubers and the productivity of potato wart cultivator differentiators in vitro.&#x0D; Methods. The research was conducted in 2019—2020 in the Laboratory of Biotechnology for Agricultural Crops of Ukrainian Plant Quarantine Research Station Institute of Plant Protection of NAAS. Derivatives of ammonium salts of dihydropyrimidine were synthesized at the Department of Organic Chemistry Institute of Biology, Chemistry and Bioresources of Chernivtsi National University. The material for the research was potato wart cultivar-differentiators of different maturity groups Slovyanka (medium ripe), Chervona Ruta (medium late). The plants were grown on Murashige-Skuga nutrient medium with addition of ammonium salts of dihydropyrimidine at different concentrations (0.1; 0.2; 0.3 mg/l) in a cultivation room at 16-hour photoperiod with a light intensity of 2000—2500 lux, temperature 22—25°C and humidity of 60—80%.&#x0D; Results. It was determined that high morphometric parameters and intensity of tuber formation of different varieties of maturity Slavyanka and Chervona Ruta in vitro culture provides the addition to the nutrient medium of ammonium salts of dihydropyrimidine with a concentration of 0.3 mg/l: plant height was 4.6 and 4.1 cm compared with the control of 4.0 and 3.7 cm, respectively. The number of internodes in both varieties were 5.3 pieces (in control — 4.5 pcs.) and the optimal number of microtubers on the 60th day — 79.1 and 81.3%.&#x0D; Conclusions. The effect study of different concentrations of ammonium salts of dihydropyrimidine on the productivity of potatoes in vitro in different varieties of maturity Slavyanka and Chervona Ruta showed that the optimal performance provides cultivation at a concentration of ammonium salts of dihydropyrimidine 0.3 mg/l: weight of the average microtuber is 22 and 195.4 mg, the mass of microtubers per plant — 185.5 and 174.4 mg, the number of microtubers per plant — 1.01 and 0.87 pcs.