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    Earthworm activity optimized the rhizosphere bacterial community structure and further alleviated the yield loss in continuous cropping lily (Lilium lancifolium Thunb.)

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    Extraction of Rhododendron arboreum Smith flowers from the forest for the livelihood and rural income in Garhwal Himalaya, India

    Study sites and samplingThe study was conducted in Garhwal region (Western Himalaya) from 2016 to 2017 at eight Rhododendron arboreum rich areas in four hill districts (Chamoli, Tehri, Pauri and Rudraprayag). Voucher specimen of Rhododendron arboreum collected and have been deposited in the Herbarium, Botany department, HNB Garhwal University (specimen no. GUH 8510)6. Identification of R. arboreum has been done through A Field Guide book authored by Rai et al.7. Since it is a wild species and flowers have been collected for our research and field study under the permission from competent authority of State Forest Department, Govt. of Uttarakhand. According to IUCN’s Red List Categories and Criteria, globally Rhododendron arboreum comes under Least Concern (LC) category8. These sites are situated between 30°08′47″ to 30°24′06″ N latitude and 78°25′05″ to 79°12′39″ E longitude with altitudes from 1820 m asl in Nandasain and 2270 m asl in Jadipani (Table 1; Fig. 1). All sites were well stocked (mean stand density ≥ 500 tree/ha) with Rhododendron arboreum trees mixed with Quercus leucotrichophora. We referred these resource rich sites as R. arboreum habitats (Table 1). Stratified random sampling method (i.e. stand density and CBH class’s strata) were carried out these eight sites. Total sampled area 0.2 ha in each site; two sample plots (size of each plot is 0.1 ha or 31.62 × 31.62 m) nested within 0.2 ha in each site were laid out for trees enumeration. Sample size (number of R. arboreum tree) for a total population in each site were 166 in Phadkhal; 110 in Khirsu; 104 in Khadpatiya; 166 in Ghimtoli; 80 in Jadipani; 74 in Ranichauri; 74 in Nandasain and 96 in Nauti. Out of the standing trees in sample plots, flower bearing trees were 96 in Phadkhal; 90 in Khirsu; 102 in Khadpatiya; 126 in Ghimtoli; 64 in Jadipani; 58 in Ranichauri; 68 in Nandasain and 82 in Nauti, and without flower or smaller trees were 70 in Phadkhal; 20 in Khirsu; 02 in Khadpatiya; 40 in Ghimtoli; 16 in Jadipani; 16 in Ranichauri; 06 in Nandasain and 14 in Nauti. The individuals of all tree species in each plot were recorded along with their CBH (circumference at breast height, 1.3 m above from the ground). Individuals were categorized as mature trees (≥ 31 cm CBH), saplings (11–30 cm CBH) and seedlings (≤ 10 cm CBH)9. Further all the tree individuals have been grouped into 8 CBH classes: (A) 5–15 cm, (B) 16–25 cm, (C) 26–35 cm, (D) 36–45 cm, (E) 46–55 cm, (F) 56–65 cm, (G) 66–75 cm, (H) 76–85 cm. Recorded data were used for the analysis of density10.Table 1 Physical characteristics of study sites in four districts of Garhwal region.Full size tableFigure 1Locations of Rhododendron arboreum study sites in Garhwal region (ARC GIS software 10.5 version was used for map preparation. The map was created by Mr. Raman Patel, Research scholar, Dept. of Geology, HNB Garhwal University, Srinagar, Uttarakhand, India).Full size imageFlower yield estimationFlower yield (kg/tree) was estimated during full bloom (flowering season/harvest season February–April 2017). In each sample plot, numbers of flower bearing trees varied from 29–63 trees/0.1 ha. At each site of 0.2 ha sample plot, total 40 trees, 05 flower bearing trees in each of the 08 CBH classes were marked for estimation of flower yield. The number of main branches, the number of sub- branches/offshoots per main branches (i.e. average per five randomly selected main branches per tree), and the amount of flower per sub-branches/offshoot (i.e. the average per five offshoots from the low, middle and upper canopy of each tree) were counted form marked individuals. This way flower yield/tree was calculated9,11,12.The flowers from all CBH classes in each site were mixed and weighted in 5 lots of 1 kg each. The number of flowers in each lot was then counted and the mean value (400.0 ± 9.56) was considered as a standard for conversion into kilograms. Based on this conversion flower yield kg/tree was obtained. Flower yield data were pooled and mean yield (kg/tree) for each CBH class (A–H) calculated. For each site, flower yield in kg/0.2 ha was obtained by multiplying flower yield/tree by the density of flower bearing trees/0.2 ha. The total yield kg/ha for each site was calculated as total yield = (yield/ha) × density of flower bear trees/ha9,11,12.Extraction/harvesting and marketing trendsFlower extraction and collection were totally dependent on market availability and accessibility of site; one of the selected sites (Ranichauri) was easily accessible, while Phadkhal, Khirsu and Jadipani were moderately accessible. Khadpatiya, Ghimtoli, Nandasain and Nauti sites were far-flung from market (Table 1). The highest extraction was recorded between second week of February and first week of April. During this period, data was obtained for three consecutive days at each site.Questionnaire based survey was carried out in selective forest fringe villages. Across the sites, total sixteen villages were selected for questionnaire survey, three villages each in Jadipani, Ranichauri, Nandasain and Nauti sites, while one village each in Phadkhal, Khirsu, Khadpatiya and Ghimtoli. In each village 15 families were randomly chosen for semi-structured questionnaire survey.Considering the market availability for trading of the R. arboreum flower products, Nandasain and Nauti sites are located nearest to local market whereas Khadpatiya and Ghimtoli sites are farthest from local market. As far as the access to resources is concerned, four sites represent open and easy access to resource and four sites represent open and moderate access of resource (Table 1). During questionnaire survey, villagers were asked about the number of persons involved in resource collection (hereafter referred as collectors), age of collectors, timing of collection (early morning and late evening) etc. Ten individuals in each group (adults and children) were randomly interviewed on their harvest load to generate data on the average collection per individual, the number of days spent in flower collection, and the total income generated through this activity.Squash/juice making factories are generally located nearby urban centers; local NGOs and small entrepreneurs are engaged in this work. These peoples purchase flower from the collectors or middleman for preparation of value product (squash). Collectors of each families (varied from n = 15 in Nanadasain to n = 31 in Jadipani) and buyers (n = 5 each site) were contacted to obtain information on the benefits accrued. The income values are given in Indian rupees (USD 1 = Rs. 68.00, 2017 exchange rates). Projections of potential (probable/-could generate) income (with flower processed into juice or squash) were made. The involvement of rural inhabitants as flowers collectors and the income that subsequently accrued (within a 10 km radius of fringe area) was also analyzed for sixteen villages across the sites. One adult member from each household was contacted in a village to collect information on involvement of flower collection/extraction.Juice/squash preparation methods and value-added productsThe collected flowers are graded for their size and healthiness and the stamens are separated from petals by laborers in the juice processing unit. Petals are cleaned washed with tap water and grinded into small pieces. The petal mass is retained in the water and then boiled for one hour. The slurry (aqueous solution) obtained in this process is left at room temperature for cooling and when it get cold, filtered through linen cloth. The filtrate solution is the pure juice of the flower. For the preparation of squash from the pure juice, about 2 kg of sugar is boiled in one liter of water. Further one liter of pure juice and a small quantity of citric acid (10 g/2 kg sugar) are added to this solution. The mixture is boiled again for 30 min and then left to cool at room temperature13. The obtained solution known as squash is then filtered through linen cloth and stored into containers and bottles for marketing. For long term storage and good test and aroma small amount of sodium benzoate and vanilla or kawra is also mixed in the squash.Cost–benefit analysis of value- added productsThe cost–benefit analysis of value added products prepared from the R. arboreum flowers was calculated in Rs./day which includes labour charges of workers involved in flower collection and materials/items required for preparation of different value added products viz: sugar, preservatives, essence, plastic containers/bottles, packaging materials etc. Labour charge was calculated on the basis of existing daily wages as per market rates. The monetary output was calculated as per the current market rates of the products (Table 2). The cost- benefit analysis of the squash product prepared from the flowers was calculated as Rs./day which includes: (i) Man days incumbent for the flowers extraction from the forest and for the preparation of squash product, (ii) Essential items such as sugar, preservatives etc. and their monetary equivalents, (iii) The total quantity of squash product and their monetary equivalents.Table 2 Market cost in rupees (Rs.) of essential commodity in the preparation of R. arboreum juice/squash in Garhwal region.Full size tableStatistical analysisData failed to meet the assumption of normality (Shapiro–Wilk test) as well as homogeneity (Levene statistic); therefore, a non-parametric test (i.e. Independent–Samples Kruskal–Wallis test) was applied for one-way ANOVA. However, to find the interaction of site and cbh on flower production (yield), the same data set was subjected to two-way analysis using univariate analysis. To find if (?) flower yield depends on tree diameter or not, data of actual cbh and flower yield per tree were used to determine a correlation (Pearson Correlation Coefficient) between them. In case of correlation found significant then regression equation was developed to predict flower production based on tree diameter. All analysis were performed using IBM-SPSS 16.0 version14.Ethics approval and consent to participateAll necessary approval, free prior informed consent, permit, and certification were secured. This was done to adhere to the ethical standards of human participation in scientific research. This study was approved by Research and Consultancy Cell (Ethics Committee) of HNB Garhwal University, Srinagar Garhwal, Uttarakhand, India. All the methods were performed in accordance with the relevant guidelines and regulations. More

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    Ovicidal activity of spirotetramat and its effect on hatching, development and formation of Frankliniella occidentalis egg

    Toxicity of spirotetramat to F. occidentalis eggsThe indoor toxicity of spirotetramat to 0-h-, 12-h-, and 24-h-old eggs of F. occidentalis using egg dipping method and leaf dipping method was shown in Table 1 after egg hatching was observed for 144 h. The results suggested that the LC50 value gradually decreased as the egg age increased. The two methods have the same trend. And in the leaf dipping method, according to the confidence limits analysis, 0-h-old eggs are significantly more sensitive to spirotretramat than 24-h-old eggs.Table 1 Toxicity of spirotetramat to F. occidentalis eggs.Full size tableEgg external shape observationsExternal morphology of normally developed isolated 0-h-old eggs of F. occidentalis in the control treatment (Fig. 1a) were compared with those treated with spirotetramat (Fig. 1b, c). After spirotetramat treatment, some eggs appeared darker, yellowish-brown, and with embryonic development abnormalities (Fig. 1b); some of the egg embryo cells treated with spirotetramat appeared atrophied, and there were obvious gaps between the outer and the inner egg embryo cells, compared with the control treatment (Fig. 1c). Some eggs ruptured at the top after the egg shell was treated with spirotetramat, and the internal egg embryo cells flowed out, thus failing to form a complete embryo (Fig. 1d); the full egg embryo cells formed a control treatment.Figure 1The effect of spirotetramat on external morphology of isolated 0-h-old eggs of F. occidentalis. (a) 0-h-old isolated eggs of normal developing thrips; (b) yellowish-brown, developmentally deformed eggs after spirotetramat treatment; (c) eggs with shrunken oocytes after spirotetramat treatment; (d) eggs with apical rupture of the eggshell after spirotetramat treatment.Full size imageThe effect of spirotetramat on external morphology of live F. occidentalis 0-h-old eggs (Fig. 2b, c) was compared with normal development in the control treatment (Fig. 2a). Similar to the effect on external morphology of isolated 0-h-old eggs of F. occidentalis, the eggs treated with spirotetramat also showed abnormal embryonic development (Fig. 2b), egg embryo cell atrophy (Fig. 2c) and the phenomenon of rupture of the egg shell and outflow of embryo cells.Figure 2The effect of spirotetramat on external morphology of living 0-h-old eggs of F. occidentalis. (a) External morphology of live 0-h-old eggs of normal developing thrips; (b) developmentally deformed eggs after spirotetramat treatment; (c) eggs with shrunken oocytes after spirotetramat treatment; (d) eggs with apical rupture of the eggshell after spirotetramat treatment.Full size imageCompared with the control (Fig. 3a), the isolated 24-h-old eggs treated with spirotetramat (Fig. 3b) did not show obvious external morphological differences. After spirotetramat treatment, the eggs were still white and plump and with no embryonic deformities, egg cell atrophy or egg shell rupture, and could still develop normally. There were clear red eye spots on the head end, and embryo movement was clearly seen under the super-depth microscope (Fig. 3b). Similarly, the live 24-h-old eggs in the control treatment (Fig. 4a) and those treated with spirotetramat (Fig. 4b) showed no obvious external morphological differences, and the eggs developed normally.Figure 3The effect of spirotetramat on external morphology of isolated 24-h-old eggs of F. occidentalis. (a) Normally developing 24-h-isolated eggs in the control group; (b) 24-h-old eggs after spirotetramat treatment.Full size imageFigure 4The effect of spirotetramat on external morphology living of 24-h-old eggs of F. occidentalis. (a) Normally developing 24-h-live eggs in the control group; (b) live 24-h-old eggs after spirotetramat treatment.Full size imageEffect of egg hatchingThe 0-h-old eggs of F. occidentalis treated with spirotetramat did not hatch normally, and the mortality rate was 100% (Fig. 5). Among them, 77 eggs eventually showed rupture of the egg shell, the internal egg embryo cells flowed out and they did not hatch; 23 eggs showed no changes in external morphology, but did not hatch after continuous observation for 144 h, and showed no developmental phenomena such as embryo movement under a super-depth microscope, which was regarded as egg death. In the control treatment, 96 eggs hatched normally, and only six eggs did not rupture but did not hatch normally and were considered dead.Figure 5Effect of spirotetramat on hatching rate of F. occidentalis 0-h-old eggs.Full size imageThere was no significant difference between the 24-h-old eggs of F. occidentalis treated with spirotetramat and the control treatment. After spirotetramat treatment, 93 eggs hatched normally, and the shells of seven eggs were not ruptured (Fig. 6). Any eggs not hatched after 144 h of continuous observation were considered dead. In the control treatment, 95 eggs hatched normally and five eggs did not rupture but did not hatch normally, and so were considered dead.Figure 6The effect of spirotetramat on hatching rate of F. occidentalis 24-h-old eggs.Full size imageSEM observationsThe F. occidentalis eggs in the control treatment were kidney-shaped, with regular egg morphology, smooth surfaces and no folds or protrusions (Fig. 7a). At 24 h after spirotetramat treatment, part of the egg shells treated with spirotetramat had fallen off the chorion, and the embryonic material was exposed (Fig. 7b). The surface of the egg shell was uneven and severely wrinkled (Fig. 7c). The pores of some eggs treated with spirotetramat were sunken down and shrunken (Fig. 7d). Spirotetramat treatment of 0-h-old eggs affect clearly egg shells, resulting in shrinkage of egg shells, ovarian depression and egg malformations, and destroyed the egg shell structure. Thus, normal embryonic development was affected, and disrupted normal hatching.Figure 7The effect of spirotetramat on the surface of egg shells of F. occidentalis 0-h-old eggs. (a) 0-h-old eggs in the control treatment; (b) eggs shells were shed 24 h after treatment with spirotetramat; (c) the surface of the egg shell was uneven and severely wrinkled; (d) the pores of some eggs were sunken down and shrunken after treatment with spirotetramat.Full size imageThe shells of eggs treated with spirotetramat (Fig. 8b) showed no significant difference compared with controls (Fig. 8a). The eggs of the two groups of F. occidentalis were regular in shape, with smooth surfaces and without folds or protrusions. Thus, development of 24-h-old eggs showed some resistance to spirotetramat. Spirotetramat did not destroy the egg shell surface structure of 24-h-old eggs, indicating a high resistance to spirotetramat.Figure 8The effect of spirotetramat on the egg shell surface of F. occidentalis 24-h-old eggs. (a) 24-h-old eggs in the control treatment; (b) 24-h-old eggs in the spirotetramat treatment.Full size imageTEM observationsThe TEM observations showed that the egg structure of the control treatment was complete, the protoplasm and yolk were clearly observed inside the egg and the yolk was packed in the void of the protoplasm network (Fig. 9a). The egg shell structure was clear, and the outer and inner egg shell were clearly observed, as was the yolk membrane and the dense layer structure (Fig. 9c). Eggs treated with spirotetramat were flocculent, and no clear internal material was observed. The protoplasm and yolk structure were blurred, and flocculation in the protoplasm appeared to agglomerate and form blocks (Fig. 9b). The egg shell structure was unclear, and no clear outer egg shell, inner egg shell, yolk membrane and lamellar structures were observed. The egg shell was also filled with many flocs (Fig. 9d).Figure 9The effect of spirotetramat on the structure of F. occidentalis 0-h-old eggs. (a) and (b) 0-h-old eggs in the control treatment; (c) and (d) 0-h-old eggs in the spirotetramat treatment.Full size imageEffect on embryonic developmentThe initial eggs of the control group were kidney-shaped, white and full of vitellin (Fig. 10a). After 12 h of development, the eggs were larger and of oval shape (Fig. 10b). After 24 h of development, the egg had increased in volume, a partially transparent region appeared in the embryo and the embryo had transparent top follicles (Fig. 10c). After 36 h of development, some yolk granules disappeared and eggs became smooth and translucent (Fig. 10d). After 48 h of development, the insect outline was visible within the egg, a pair of antennae were visible on the head and a red eye point was clearly observed on the head during the blastokinesis phenomenon (Fig. 10e). After 60 h of development, embryo color deepened, the eye point was clearer and the head, femur, tibia and tarsus were clear (Fig. 10f). After 72 h of development, the egg shell began to break at the head, the tail constantly jittered, internal fluid flowed and the larva hatched from the top of the egg (Fig. 10g).Figure 10The embryonic development process of control 0-h-old eggs of F. occidentalis. (a) Control initial eggs; (b) eggs after 12 h of development; (c) eggs after 24 h of development; (d) eggs after 36 h of development; (e) eggs after 48 h of development; (f) eggs after 60 h of development; (g) eggs hatching as larvae after 72 h of development.Full size imageEggs of F. occidentalis were initially white, kidney-shaped and full of vitellin (Fig. 11a). Following treatment with spirotetramat, after 12 h of development, the eggs became large and oval, and the embryo was a pale brown color (Fig. 11b). After 24 h of development, color of the egg deepened to dark brown. There was a gap between the egg and the egg shell, and a small amount of spillage appeared at the end of the egg (Fig. 11c). After 36 h of development, the egg shell ruptured, material flowed out of the egg and embryo development did not proceed (Fig. 11d).Figure 11Effects of spirotetramat on development of 0-h-old eggs of F. occidentalis. (a) Frankliniella occidentalis initial eggs; (b) eggs developing 12 h after spirotetramat treatment; (c) eggs developing 24 h after spirotetramat treatment; (d) eggs developing 36 h after spirotetramat treatment.Full size imageIn the control treatment, the egg volume increased at 24 h, the embryo had a partially transparent area and there was a transparent follicle on the top of the embryo (Fig. 12a). After 12 h of development, some of the yolk particles disappeared and the egg body was smooth and translucent (Fig. 12b). After 24 h of development, the body outline, a pair of antennae and red eye spots were visible, and there was obvious embryo movement (Fig. 12c). At 36 h of development, the head, leg segments, tibia and tarsus were apparent (Fig. 12d). After 48 h of development, the embryo moved violently, internal body fluid flowed and the larva was ready to hatch (Fig. 12e). After 60 h of development, the larva emerged from its shell (Fig. 12f).Figure 1224-h-old eggs embryo development of F. occidentalis in control treatment. (a) Control 24-h-old eggs; (b) eggs after 12 h of development; (c) eggs after 24 h of development; (d) eggs after 36 h of development; (e) eggs after 48 h of development; (f) eggs hatching as larvae after 60 h of development.Full size imageThe 24 h old eggs of F. occidentalis showed enlarged volume, and there were transparent follicles on the top of the embryo (Fig. 13a). After 24 h eggs were treated with spirotetramat, they developed for 12–36 h, and the developmental status was the same as that of the control. The embryos developed normally, and there was no egg body discoloration or egg shell rupture (Fig. 13b–d). After 48 h of development, hairy scales appeared on the surface of the egg shell, and the egg body turned yellowish-brown in color, but the egg shell was not broken and no internal material overflow was seen (Fig. 13e). After 60 h of development, larvae hatched normally (Fig. 13f).Figure 13The effect of spirotetramat on embryonic development of F. occidentalis 24-h-old eggs. (a) Frankliniella occidentalis 24-h-old eggs; (b) eggs developing 12 h after spirotetramat treatment; (c) eggs developing 24 h after spirotetramat treatment; (d) eggs developing 36 h after spirotetramat treatment; (e) eggs developing 48 h after spirotetramat treatment; (f) eggs hatching as larvae after 60 h of development.Full size image More

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    High canopy cover of invasive Acer negundo L. affects ground vegetation taxonomic richness

    1.Vinogradova, Y. K., Mayorov, S. R. & Khorun, L. V. Chernaya kniga flory Sredney Rossii (Chuzherodnye vidy rasteniy v ekosistemakh Sredney Rossii) (The Black-book of the flora of the Middle Russia (Alien species in the plant communities of the Middle Russia). (GEOS, 2010).2.Straigytė, L., Cekstere, G., Laivins, M. & Marozas, V. The spread, intensity and invasiveness of the Acer negundo in Riga and Kaunas. Dendrobiology 74, 157–168 (2015).Article 

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