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RearingAll mosquitoes used in these experiments were derived from a laboratory-reared colony of An. stephensi is initially established (six generations) in University College Agriculture, University of Sargodha. Uninfected mosquitoes were maintained in the laboratory; in gauze-covered boxes (30 cm wide × 30 cm high × 50 cm deep) under control condition 27 ± 2 °C temperature and 75–80% relative humidity. Auto ON/OFF switches with the timer were used to break the scotophase (dark) period in the control conditions of the laboratory with the light: dark cycle set to 12:12 h48. A 10% fructose solution supplemented with 0.05% para-minobenzoic acid (PABA) was provided to mosquitoes. The adult mosquitoes were reared on blood provided via an in situ electronically derived artificial membrane feeder, set at 37 ± 1 °C, and offered twice a week, and offered twice a week given their need for another blood meal approximately 5–6 h after the first49. Multiple blood feeding is vital for Anopheline species as it has been demonstrated as influencing reproductive behavior50. Oviposition cups were provided two days following the second blood meal. The larvae were reared under laboratory conditions described above and provided a certified Laboratory Rodent Diet (LRD) Lab Diet 500151.Fecundity and fertilityTo determine differences in fecundity (number of eggs) and fertility (percentage of fertile eggs) in An. stephensi mosquitoes, cages of mosquitoes were provided ABO blood groups and control (distilled water) via artificial membrane feeders (as described above for mosquito rearing). The blood was obtained from the blood bank of DHQ (one batch of each blood group was used for each replication), Chakwal Punjab, Pakistan. A total of 10 replicate cages were used for each blood type and control, so 10 batches of each blood group (ABO) (just to reduce the potential individual to individual variation) were obtained from the DHQ. After each replication along with the new batch of each blood group, a new strain of mosquitoes was used just to reduce the learning behavior of mosquitoes. Feeding success was determined by calculating the percentage of fed mosquitoes, and also, the numbers of fully engorged female mosquitoes were recorded.To determine fecundity, females from each blood group were removed, killed, and dissected under a microscope, and the numbers of eggs per female were counted 60 h post-blood-feeding. Additionally, to determine oviposition and larval development, 40 fully fed female mosquitoes were caged in one of three replicate glass cages with gauze (25 cm wide × 25 cm high × 25 cm deep) and provided wet filter papers were placed for egg-laying. The total number of eggs was counted after every 12 h from 48 h until 96 h post-blood-feeding under a light microscope. The total numbers of eggs/40 females/box for each human blood group for 10 replicates were calculated.For fertility estimation, an additional 40 gravid An. stephensi from each treatment and replication, including the control group, the females were gently transferred to the cages with triangular Whatman filter paper No. 1 using the mouth aspirator. The egg laid in each experimental and control group was reared in plastic trays filled with distilled water. The numbers of hatched larvae were recorded for a fertility test. While the eggs that could not be hatched into larvae up to day 7 were considered as infertile. The number of fertile and infertile eggs was recorded from all experimental and control cages.The collected eggs from each experimental box were placed into the plastic trays (24 cm wide × 12 cm high × 7 cm deep) with water, and the development of mosquitoes was observed until adult mosquitoes had emerged from all pupae for each blood group. The water in these plastic trays was maintained at a constant level throughout immature mosquito rearing. The larvae were fed a certified Laboratory Rodent Diet (LRD) Lab Diet 500151. The rearing was done according to the standard mass rearing of Anopheles techniques52. Pupae were counted and removed from the tray and placed in cages according to each human blood group type fed to allow emergence, and the percent of male and female mosquitoes was recorded. Adult mosquitoes were maintained on a 10% fructose solution supplemented with 0.05% para-minobenzoic acid (PABA) but were not provided with a blood meal. The mortality of adult mosquitoes was recorded daily until total mortality reached 100%.Digestibility testsThe precipitin and benzidine tests were used to test the effect of human blood groups ABO (on the rate of digestion in mosquitoes). The experiment was conducted in controlled laboratory conditions where the temperature, humidity, and day and night periods were maintained as described above. Mosquitoes (10 mosquitoes were used for each blood group and the same experiment was repeated 10 times) that had not been fed previously on either a sugar solution or blood were used in experiments. Mosquitoes were provided one of four different human blood group types, as previously described. After feeding the female were kept in the same boxes without any further food and water, and boxes were placed in an incubator where the temperature and the relative humidity was at a constant level (28 ± 2 °C and 80 ± 5%). The engorged female adult mosquitoes were killed at 8 h intervals, rubbed over the filter paper53, and the filter papers were placed inside the refrigerator until the test could be conducted. Approximately 48 female mosquitoes were used in each boxed marked for each blood group. The rate of blood digestion in the engorged blood was classified according to the Sella scale, following Detinova et al.54.To perform the precipitin test, the physiological saline and the filter paper smears were extracted in a small capillary tube. The specific antiserum was also extracted in the same capillary tube at the end; the change in color, clumping, and cloudiness of the solution indicates the presence of human blood in the tissue smears55. The collected material was heated in a steam oven for 10–12 min to apply the benzidine test at 108–110 °C. The test was used to check the traces of iron porphyrins in the abdomen of mosquitoes.Effect of blood groups on oogenesisTo test the blood-specific effects on the development of the ovaries of An. stephensi, the ovaries of fully fed female mosquitoes were collected separately from the box of each blood group 36 h post engorgement. For scanning electron microscopy (SEM), the whole female mosquitoes were selected from each box of every blood group separately (10 females for each blood group ABO). In preparing specimens for the scanning electron microscope, the process is divided into two fixations, the primary and the secondary fixation. For the primary fixing process, the 2.5% glutaraldehyde in 0.1 M cacodylate buffer was used for the period of 2 h, followed by the three consecutive washing with the same buffer for the 30 min. While for the secondary fixation process, 1% osmium tetroxide was used for the 2 h. Then the samples were rinsed for the final time with the 0.1 M cacodylate buffer three times for 30 min.The ovaries were dehydrated by using the graded series of acetone (50%, 70%, 80%, 90%, and 100%). The dehydrated ovaries were then transferred to the critical point drying apparatus. The recommended quantity of acetone solution was also poured into the drying chamber to avoid over-drying. Liquid nitrogen was also added into the drying critical point drying chamber. The CO2 and acetone were allowed to be mixed freely; the same process was repeated eight times to confirm the drying of the specimen. The dried mosquito specimens were mounted over the stubs, and the specimens tubes were coated with a thin layer of silver. Gold-spotted SCD005 was used, and then the samples were photographed with SEM.Electroantennography (EAG)To measure the response of mosquitoes to each human blood group type, EAG recordings from one antenna of An. stephensi female mosquito was made. Unfed female mosquitoes were anesthetized by the use of CO2 and were permanently fixed with the reference electrode by the use of spectra 360 electrode gel. It was made sure that the mosquito was completely immobile except for the antennae. The tips of the antennae were pressed into the small drop of electrode gel on the recording electrode. Both of the electrodes are silver wires coated with silver chloride with a diameter of 0.2 mm. The experimental preparations were done in continuous airflow (600 mL/min, 1.5 m/s) by the Teflon tube of 0.7–0.8 cm diameter, containing about 100 mL/min dry air and the 600 mL/min moist air passed through the charcoal filter. At this stage, little modification was done in the structure of the electroantennogram, and an artificial blood feeder of mosquitoes with the membrane was attached to the system.The blood feeder was packed in a glass jar through which continuous airflow was passed, and this air flow ends as stimuli near the mounted mosquito. The diameter of the glass tube was 0.5 mm, and the flow of air was controlled by using an ON/OFF switch; three bursts of 0.5 s of air from the blood jar were provided as a stimulus to host-seeking mosquitoes. All the blood groups were tested systematically together with the control group. The amplifier amplified the generated signals while the well-known software decoded the recordings (EAG 2000, Syntech, Hilversum, and the Netherlands). All of the test blood groups were also dissolved separately in tetryl-butyl-methyl ether (MTBE), and about 30 uL of this test solution was applied onto a piece of filter paper (1.5–2 cm). About 20 min was given to the TMBE solution to evaporate from the filter paper leaving behind the blood; then, this piece of filter paper was placed into the Pasteur pipette. In the case of the control treatment, distilled water was used, and the same treatment was applied with the distilled water for the test compounds. The stimulus controller C5-01/b, Syntech, was used to inject the odor cues originating from the treated filter paper in the Pasteur pipette into the humidified and filtered air stream directed towards the antennae of immobilized mosquitoes. Olfactory stimuli were tested randomly against different mosquito specimens with a total of five specimens exposed to each of the human blood type groups and control.To minimize the chances of error and to test the electrophysiological activity of the stuck female mosquito, lactic acid, 1-octen-3-01, and isovaleric acid were used as known stimulants41,56. After that, each blood group was replicated three times to record the activity of the olfactory neurons of antennae. All the treatments of blood groups were tested randomly, and a regular interruption of control stimulus (0.1% lactic acid) was done. The regular interruption of the control stimulus was used to control the activity of the antennae. All the stimulants were expressed as a mean percent response to the control treatment. The response of different female mosquitoes to human blood groups was indicated as a mean percent response. The results were analyzed by the use of the Student’s t-test.Wind tunnel bioassaysWind tunnel bioassays were used to determine the response of An. stephensi to four human blood groups (A, B, AB, and O) and control stimuli (distilled water). Wind tunnel bioassays have been used to evaluate the response of Ae. aegypti, Cx. quinquefasciatus and Cx. nigripalpus towards the blood volatiles44. A dual choice wind tunnel was converted into a “five-choice” tunnel with all five glass tubes having glass jars at their end with openings to accommodate an artificial blood feeder. A continuous flow of warm water ensured the blood remained in liquid and produced its specific smell.A batch of approximately 100 female mosquitoes was released at the downwind end of the tunnel in the air stream coming from the five upwind end chambers. After 30 min, the numbers of mosquitoes in each of the five glass jars were counted. The mosquitoes were then sent back towards the downwind end of the wind tunnel, and the positions of odor cues were changed, including the control. Before the second time release, the fresh air was passed through the tunnel. Again the mosquitoes were released from the releasing box, and the response of the mosquitoes towards the new cues and the number of mosquitoes in each chamber at the upwind end was counted after 30 min; the same process was repeated, and for the third time with randomization. The same process was repeated 10 times with each blood group and with a new batch of mosquitoes each time.To test the response of female mosquitoes towards human-emitted olfactory cues, an olfactometer was used in previous studies41,43. The olfactometer test was conducted in the control room; the temperature was 27 ± 2 °C with 70–80% relative humidity. The optimum activity of the An. stephensi was observed late at night, so the experiment was conducted at 2–6 AM57.Steel balls rubbed in the hands of persons (ten volunteers per blood group) of having ABO blood groups along with the few drops of blood group-specific sweat were placed in the glass jars at the upwind end of the olfactometer. Approximately 100 female mosquitoes were released at the downwind end of the olfactometer from the releasing cage. After 30 min, the total number of mosquitoes in each box at the upwind end was counted, including the control. After that, the mosquitoes were returned to the releasing cage; then, the positions of steel balls at the upwind end of the glass jar of the olfactometer were changed randomly to decrease the biases from the data. To remove the smell of a sweat from the olfactory tube after cleaning, the fresh air was passed for about 10 min continuously. Mosquitoes were then again allowed to enter into the olfactometer, and after 30 min, the total number of mosquitoes was counted. The same process was repeated for the third time. The same experiment was repeated 10 times with different persons and mosquitoes to decrease the chances of error. A new batch of mosquitoes was selected for each replication.Statistical analysisThe mean number of eggs of An. stephensi were evaluated with the help of a linear model (ANOVA) and Tukey’s test on Minitab® software (12.2, version, Minitab). Before performing the ANOVA, with the help of angular transformation (arcsine √x), egg viability was also transformed58 for fertility. Data obtained were analyzed using R 3.2.2 software. The Shapiro–Wilk normality test was carried out, which showed that the data were not normally distributed. Hence, the Kruskal–Wallis Chi-square test was used to compare the averages of the responses of An. stephensi in relation to blood-group treatments.Ethics statementAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed. More

EDITORIAL
21 December 2021

Sustainability at the crossroads

A look back at 2021 through the Sustainable Development Goals.

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A medical worker observes people with COVID-19 inside a makeshift care facility at the Commonwealth Games Village in New Delhi in May 2021.Credit: Getty

There were high hopes for 2021. The year promised progress on the push for sustainable development after months of pandemic-induced delays and uncertainty. We heard ambitious talk of a ‘green recovery’, and world leaders were due to gather for meetings of the United Nations conventions on biological diversity and on climate to set future agendas.How did the year’s sustainability debates evolve? We take a look through Nature’s science lens.2021: a year of multiple crisesAs 2021 draws to a close, the world is facing numerous crises. The COVID-19 pandemic is far from over. A year after the first vaccines began to clear regulatory hurdles, the emergence of the SARS-CoV-2 Omicron variant is challenging the fragile and unequal gains in bringing the virus under control. Progress is slow on mitigating and adapting to climate change, protecting biodiversity and ending hunger — parts of the Sustainable Development Goals (SDGs), the United Nations’ flagship plan to end poverty and promote a healthier planet by 2030. The plan, already off track before the pandemic, has been all but derailed by COVID-19.
More floods, fires and cyclones — plan for domino effects on sustainability goals
Nature has argued1 that the setback requires a more rapid response by the researchers who are writing the latest UN Global Sustainable Development Report — the scientific input to the SDGs, which runs on a four-year cycle. But attempts to feed science into policy have come up against strong barriers. Democracy and multilateralism are in retreat, undermining the commitment needed to make progress on sustainability goals. Still, this should not be a reason to disengage. On the contrary, researchers generally need to redouble their efforts.Fighting the climate crisisEarly November was marked by a momentous climate summit, the 26th UN Climate Change Conference of the Parties (COP26) in Glasgow, UK. For the first time, the final agreement included mention of a phase down of coal-fired power, although phase out was the original aim. It also called for the ending of some public subsidies for other fossil fuels — one of the biggest financial barriers to the shift to renewable energy. More than 100 countries pledged to cut methane emissions, flagged for their role in global warming in the latest report from the Intergovernmental Panel on Climate Change (IPCC)2. Richer nations committed to doubling their funding by 2025 to help low- and middle-income countries (LMICs) deal with the damage already caused by climate change, and they agreed to set up an office to research a long-proposed fund to compensate LMICs for that damage.But even if the pledges announced are implemented, temperatures are still projected to rise to a catastrophic 2.4 °C by 2100. And below the surface lay disagreements on definitions and the detail of implementation. And this is where research must continue to offer essential input. ‘Net-zero’ is one example. There is no agreed definition or measure of it, and without this, it’s impossible to know whether pledges will actually stop global warming. There is also no agreed definition of climate finance for LIMCs. This means that richer countries can make up their quotas with loans or official development aid that links to climate change only indirectly. Arguments have persisted for years over the funding promised more than a decade ago — what has been disbursed and who owes what — and this has undermined trust and has cast a shadow over negotiations, including those in the lead-up to the Glasgow meeting.

Protesters hold a ‘Biodiversity Emergency’ banner during the demonstration outside the Bank of England in London in November 2021.Credit: Vuk Valcic/SOPA Images/LightRocket/Getty

Elusive biodiversity protectionJust days before COP26, at a separate COP hosted by China in Kunming in Yunnan province, governments debated measures to protect the diversity and richness of plant and animal species. In the first sessions of a two-part UN summit on biological diversity, due to conclude in May 2022, discussions centred on a widely supported target to protect 30% of the world’s land and sea areas by 2030 — up from the previous ‘Aichi target’ of 17%. Among other targets under debate was the provision of greater financial support to low-income countries to preserve biodiversity.
The world’s species are playing musical chairs: how will it end?
Progress on biodiversity protection has proved elusive since the first ‘Earth Summit’ in Rio de Janeiro in 1992. The Kunming summit ended with a modest boost in funding for projects that help to preserve biodiversity — unlike climate change, funding for biodiversity comes mostly from the public sector. We argued that these contributions should be given as grants, rather than loans that saddle poor countries with debt3. This is now more important than ever, as the pandemic piles perilous debt on the developing world.Protecting biodiversity goes hand in hand with managing land and water resources sustainably, and in this way aligns with tackling climate change. And if nature continues to degrade, sooner or later economic output will suffer. This link is captured by debates over assigning monetary and other values to ecosystems, an idea no longer theoretical or controversial. In March, we welcomed a move by members of the UN Statistical Commission to finalize a set of principles that will help national statisticians record ecosystem health and work out payments for ecosystem services4.

Icebergs that calved from the Sermeq Kujalleq glacier in Greenland this year help mark one of Greenland’s biggest ice-melt years in recorded history.Credit: Mario Tama/Getty

Revamping food systemsLike biodiversity protection, the world’s food system needs fixing. One in ten people is undernourished and one in four is overweight. The number of people going hungry is rising fast, a trend fuelled by the pandemic. Nature’s coverage emphasizes the fact that science needs to guide the transformation of the food system. The task is challenging, because food spans many disciplines. We have yet to pin down what diets that are both healthy and sustainable should look like. And an IPCC-like system of scientific advice to inform policymaking has so far been missing from food and agriculture.
What humanity should eat to stay healthy and save the planet
That changed in September, when António Gutteres, the UN secretary-general, convened a controversial but historic Food Systems Summit. A group of scientists was tasked with ensuring that the science underpinning the summit was robust, broad and independent. Writing in Nature, this scientific group issued seven priorities for research, among them a greater focus on sustainable aquatic foods5. Soil-based agriculture tends to dominate discussions on food, with ‘blue foods’ — organisms such as fish, shellfish and seaweeds — rarely considered.Nature joined the scientific group’s call to argue that it’s time to change that (see go.nature.com/3e3ss6r). We published the Blue Food Assessment — the first systematic evaluation of how aquatic food contributes to food security — which explores how research can help transform the global food system. This work also shows some pitfalls of a greater reliance on blue foods without sustainable management, as a rapidly increasing demand for fish adds to risks for coastal ecosystems and the people of coastal communities.

Volunteers prepare meals for distribution in the Paraisopolis favela in São Paulo, Brazil, in March 2021Credit: Jonne Roriz/Bloomberg/Getty

Strong moves from the UN’s centreThe year 2021 also saw various arms of the UN consider how their own governance needs to respond and adapt to changing times. Guterres is set to appoint a new board of scientific advisers to his office, a decision that Nature welcomed6. The decision is part of the organization’s 25-year vision, laid out in the secretary-general’s report, Our Common Agenda (see go.nature.com/3egrudq), in September. Specialized agencies also needed to stocktake. Over the fifty years since its founding, the UN Environment Programme has pushed important initiatives that bring science into ‘green’ policy — co-founding the IPCC, for one — and we urged it to do more to bring together researchers from across environmental sciences to tackle interconnected challenges7. Nature also urged the International Monetary Fund’s shareholders to lend money to strengthen universities, so that science can better work towards global goals8.
The broken $100-billion promise of climate finance – and how to fix it
The right moves at the top echelons of global governance matter – but support for science and collaboration within and between countries matter just as much. In some ways, LMICs are leading the way. A 700-page report by the UN science and cultural organization UNESCO is a first attempt to understand the impact of the SDGs on research priorities9. It found that, unlike richer nations, lower-income countries’ share of research publications jumped in areas such as photovoltaics and climate-resilient crops. Individual countries need to do better to boost innovation, but collaboration will prove crucial. We need look no further than the pandemic for examples of how researchers working across borders, cultures and disciplines can benefit science and society.Collaboration and inclusionWe need — and can — do better on collaboration. Global problems need diverse teams to help navigate social and geopolitical challenges. Our COVID-19 coverage comes with a host of inspiring stories of scientists joining forces to tackle the crisis. It serves as a reminder of what can be done. But it’s not easy. Collaboration means spending less time achieving metrics of performance and more time nurturing relationships. Link-ups between science and industry suffer without rules around data ownership and intellectual property. And mounting geopolitical tensions, particularly between the United States and China, are limiting exchanges of people and knowledge10.
How the COVID pandemic is changing global science collaborations
The benefits of international research are worth the effort for both LIMCs and wealthy nations. But collaborations often come with concerns over equity and who benefits. Concerns over inclusion extend to policy forums too. At COP26, Nature found that researchers were frequently prevented by the organizers from accessing negotiations. Representatives from civil society and the global south also complained of exclusion. That experience must not be repeated. We’ve also argued that forums such as the G7 group of wealthy nations and the World Health Organization should regard emerging economies as equals. And UN bodies that solicit scientific input need to reach out beyond their usual expert networks to involve under-represented communities. The Food Systems Dialogues (see go.nature.com/3ykm2ye) could be a model: this initiative has engaged hundreds of participants across six continents since 2018, becoming an official mechanism to build international consensus at the UN food summit.An eye on the futureLooking ahead to 2022, we’re keeping our finger on the pulse. Nature will maintain a focus on climate, global health and sustainability. We expect more attention to the food crisis and climate-related migration, and more debate on solutions and trade-offs tied up with the energy transition.
Why fossil fuel subsidies are so hard to kill
The fallout from the pandemic will be a key focus. It includes the burden of disability from long COVID, lost ground in the fight against polio, malaria, tuberculosis and HIV, the lifelong impact of the loss of education for millions of children, and rising violence against women and girls. As economies struggle to get back on their feet, the financing of sustainability goals is an urgent issue that needs resolving. Researchers should also work towards resolving some of the long-standing tensions between climate, biodiversity conservation and food provision.The SDGs remain a holistic framework for guiding priorities for sustainable development. In the shorter term, we look to next year’s conclusion of the biodiversity summit, and the climate summit in Cairo. And we stand ready to support science as it responds to global challenges by engaging with policy and the public.

Nature 600, 569-570 (2021)
doi: https://doi.org/10.1038/d41586-021-03781-z

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Plant height (cm)Plant height of black cumin as affected by moringa leaf extract applied at various growth stages is reported in Table 1. Both concentrations of moringa leaf extract significantly affected plant height of black cumin. All growth stages also showed statistically significant results. Mean comparison of control vs treatments and water spray vs rest were also found significant for plant height (cm) of black cumin. Whereas, interaction of moringa leaf extract concentrations and growth stages remained non-significant. With increase in interval of spraying moringa leaf extract, plant height enhanced and thus taller plants (68.15 cm) were recorded when moringa leaf extract was sprayed at stage-7 (40 + 80 + 120 days after sowing), followed by (65.15 cm) stage-4 (40 + 80 days after sowing), while lower plants height (47.45 cm) was recorded in stage-3 (120 days after sowing). The use of moringa leaf extract during critical vegetative development phases increased the black cumin crop’s plant height. Similar results were recorded by Abbas et al.14 that moringa leaf extract enhanced plant height and improved fresh and dried weight of wheat root when compared to control. Taller (62.2 cm) plants were recorded in 20% moringa leaf extract sprayed plots followed by (55.8 cm) 10% moringa leaf extract. Spraying moringa leaf extract on a variety of field crops can boost plants and increase vegetative development15.Table 1 Plant height (cm), number of branches plant−1 fixed oil content (% vw−1) and essential oil content (% vw−1) of black cumin as affected by moringa leaf extract applied at various growth stages.Full size tableBranches plant−1
Branches plant−1 of black cumin were significantly influenced by moringa leaf extract concentrations, stage of application as well as their interaction (Table 1). The planned mean comparison of control vs rest and water spray vs rest were also found significant for branches plant−1. The unsprayed against sprayed treatments of moringa leaf extract showed that in unsprayed plots number of branches plant−1 (39) were less than plants sprayed with moringa leaf extract (61.19). Highest number of branches plant−1 (62.19) were observed 20% moringa leaf extract treated plots. These results are in agreement with Mahmood16 who found that foliar application of MLE contains an adequate amount of stimulating substances that promote cell division and enlargement at a faster rate. Zeatin, a growth hormone found in moringa leaf extract, encourages the growth of lateral buds, which leads to an increase in the number of branches. After pounding 100 g of Moringa leaves in 8 L of water, foliar spray of moringa leaf extract enhanced branches plant−1 in okra17. More number of branches plant−1 (70.66) were attained in plots sprayed with moringa leaf extract at growth stage 7 (40 + 80 + 120 days after sowing), followed by growth stage 4 (40 + 80 days after sowing). The effect of the application of MLE at the rate of 20% at 40 days’ interval increased the number of branches and this may be because of the abundant supply of macro and micronutrients and growth hormones. The result of yield parameters revealed that the yield increased as the frequency of moringa leaf extract increased. This is because hormone enhances formation and development of flowers and ripening of fruits. Hormones also enhance growth and yield by altering photosynthetic distributive pattern within the plants. The findings were also in line with that of Manzoor et al.18 who found that an aqueous extract of moringa significantly influence yield and yield components such as number of branches, number of fruits per plant and fruit weight of tomato. The significant interaction of MLE and growth stages is presented in Fig. 1. Applying moringa leaf extract @ 20% at all growth stages enhanced branches plant−1. Maximum branches plant−1 was observed when moringa leaf extract was sprayed @ 20% at growth stage 7 (40 + 80 + 120 days after sowing) whereas, minimum branches plant−1 was recorded in plants sprayed with 10% moringa leaf extract at growth stage-3 (120 days after sowing). Moringa leaf extract (MLE) increased number of branches. Similar results were recorded by Jain et al.19), who reported MLE positively enhanced plant growth attributes of wheat. He also stated that with increasing MLE concentration and application intervals, the growth parameters such as branches plant−1 were increased in arithmetic order. Plant growth regulators are essential for controlling growth and development of plants20. These plant growth regulators increased yield by changing the dry matter distribution pattern or controlling the growth characteristics in crop plants, depending on the dosage and time of application21. In comparison to control, foliar application of moringa leaf extract resulted in a markedly higher branches plant−1. The increased number of branches plant−1 might be due to Zeatin present in moringa leaf extract, which is very effective in delaying the abscission response10.Figure 1Number of branches plant−1 of black cumin as affected by moringa leaf extract applied at various growth stages.Full size imageFixed oil content (% vw−1)Data concerning fixed oil content (% vw−1) in response to moringa leaf extract applied at various growth stages is given in Table 1 and Fig. 2. Statistical analysis of data indicated that foliar application of various concentrations of moringa leaf extract, their stage of application and interaction of concentrations and growth stages had significantly affected fixed oil content (% vw−1) of black cumin crop. The planned mean comparison of control vs rest and water spray vs rest had significant effect on fixed oil content (% vw−1). Highest fixed oil percentage (35.39%) was recorded when moringa leaf extract was sprayed @ 20%, followed by (34.06%) 10% moringa leaf extract, whereas, control (31.48%) showed lowest fixed oil %. Sakr et al.22 indicated that foliar applications of MLE significantly improved the oil percentage and yield plant−1 and feddan of geranium plants. Application of MLE at growth stage-7 (40 + 80 + 120 days after sowing) showed maximum fixed oil content percentage (37.08%) as compared to all other growth stages. Minimum fixed oil percentage was recorded in growth stage-1 (40 days after sowing). Concerning the interaction of moringa leaf extract vs application stage, highest fix oil (37.45%) was observed when moringa leaf extract @ 20% was applied as foliar spray at growth stage-7 (40 + 80 + 120 days after sowing), followed by (36.71%) moringa leaf extract @ 10% applied at growth stage-7. Lowest fixed oil percentage (31.83%) was observed in plants sprayed with 10% moringa leaf extract at stage 1 (40 days after sowing). According to Rady et al.23, biosynthesis of cytokinins promotes the movement of stem reserves to new shoots, resulting in stable plant development, the prevention of premature leaf senescence, and the preservation of more leaf area for photosynthetic action.Figure 2Fixed oil content (%) of black cumin as affected by moringa leaf extract applied at various growth stages.Full size imageEssential oil content (% vw−1)Essential oil content (% vw−1) is a vital oil component of black cumin. Moringa leaf extract concentrations and stage of their application had significant effect on essential oil content of black cumin while the interaction remained non-significant (Table 1). Application of MLE at 20% resulted in higher essential oil yield (0.38%) followed by 10% moringa leaf extract (0.37) sprayed plots. Control plots resulted in lower essential oil (0.33%) content of black cumin. Many research ventures around the world are currently focusing on increasing the biomass yield and volatile oil output of aromatic plants. Moringa leaf extract has been discovered to be an excellent bio-stimulant for enhancing not only crop growth but also yield24,25. According to Aslam et al.26, Plant treated with MLE had major impacts, including an average rise in oil concentrations. Interestingly, MLE treatment not only increased the coriander fruit yield but also improved the fruits volatile oil suggesting that MLE could be a promise plant growth promoter that improved the content of volatile oil in coriander. MLE application also positively affected the volatile oil constituents (Table 2). Increasing the volatile oil in coriander by MLE could be due to the MLE components including amino acids, nutrient elements and phytohoromes that motivate the accumulation of secondary metabolites27. The phytohormones affect the pathway of terpenoids through motivating the responsible physiological and biochemical processes28. Concerning the application stages of moringa leaf extract, higher essential oil content % of black cumin (0.42%) was observed in growth stage-7 (40 + 80 + 120 days after sowing), followed by (0.39%) growth stage-4 (40 + 80 days after sowing), whereas, lower essential oil content % (0.36%) of black cumin was observed in growth stage-1 (40 days after sowing). Plant growth regulators are essential for controlling the amount, type, and direction of plant growth, development, and yield20. These plant growth regulators increased yield by changing the dry matter distribution pattern or controlling the growth characteristics in crop plants, depending on the dosage and time of application21. Exogenous application of MLE resulted in higher yield and quality29.Table 2 Peroxidase value (meq kg−1) and Iodine value (g of I2/100 g) of black cumin as affected by moringa leaf extract applied at various growth stages.Full size tablePeroxidase value (meq kg−1)The response of MLE and stage of MLE application recorded for peroxidase value is stated in Table 2. The data depicted that moringa leaf extract concentrations, stage of application and their interaction had significant (P ≤ 0.05) variation in peroxidase value of black cumin. Similarly, when means were compared, that of control vs treatments and water spray check vs treatments were found significant for peroxidase value (%). Mean value of data indicated that highest peroxidase value (6.32%) was recorded in 20% moringa leaf extract treated plots, followed by (6.03%) 10% moringa leaf extract. While in case of application stages, highest peroxidase value (6.42%) was recorded when moringa leaf extract was applied at stage-7 (40 + 80 + 120 days after sowing), followed by (6.39%) stage-6 (80 + 120 days after sowing). Whereas lowest peroxidase value (5.73%) was recorded in plots treated with moringa leaf extract at stage-3 (120 days after sowing). Interaction of moringa leaf extract concentrations and stage of application in Fig. 3 showed that increasing moringa leaf extract concentration from 10 to 20% applied at growth stage-7 increased peroxidase value of black cumin crop. However, application of moringa leaf extract @ 10% applied at growth stage-3 (120 days after sowing) showed lowest peroxidase value. The phytohormones affect the pathway of terpenoids through motivating the responsible physiological and biochemical processes28. Our results are in agreement with the reports of Ali et al.27 in geranium and Abdel-Rahman and Abdel-Kader30 in fennel who observed that MLE application improves both the volatile oil yield and its components. The fact that MLE application improved black cumin growth and quality characters suorts the study’s hypothesis that MLE is an important plant growth enhancer. In agreement with our results, Rady and Mohamed28 concluded that MLE is considered one of the important plant bio stimulants because it contains antioxidants, phenols, basic nutrients, ascorbates, and phytohormones. Furthermore, foliar application of moringa leaf extract may have a positive effect on endogenous phytohormone concentrations, resulting in improved plant growth and quality10,37.Figure 3Peroxidase value (meq kg−1) of black cumin as affected by moringa leaf extract applied at various growth stages.Full size imageIodine value (g of I2/100 g)Data concerning iodine value of black cumin oil in response to various concentrations of MLE applied at various growth stages is given in Table 2 and Fig. 4. Statistical analysis of data indicated that both the concentrations of moringa leaf extract, stage of application as well as their interaction had significant effect on iodine value of black cumin oil. The planned mean comparison of control vs rest and water spray vs rest treatments had significant effect on iodine value. Highest iodine value (85.3) was recorded with application of moringa leaf extract @ 20% whereas, lowest (78.28) was observed in control. Regarding the stage of application, highest iodine value (87.35) was observed in plots sprayed with moringa leaf extract at stage-7 (40 + 80 + 120 days after sowing), followed by (85.61) plots sprayed with moringa leaf extract at growth stage-6 (80 + 120 days after sowing). Concerning the interaction of MLE concentrations and stage of application of MLE, highest iodine value (6.49) was observed with 20% moringa leaf extract sprayed at stage-7 (40 + 80 + 120 days after sowing) whereas, lowest iodine value was observed in plants sprayed with moringa leaf extract @ 20% applied at stage-3 (120 days after sowing). The use of plant growth regulators is very specific and depends to achieve specific results like for example; enhanced plant growth, betterment in yield and yield related attributes, and to modify the fruit and plant bio-constituents. Several previous studies reveled that MLE are enriched with many phtyo-hormones especially zeatin31. In addition to that MLEs are embedded with many essential amino acids, vitamins (A, B1, B2, B3, C and E), minerals as well as several antioxidants like phenolic32,33. This unique biochemical composition of MLE showed that they can be utilized as bio stimulant which have the potential to promote crop growth, productivity as well as quality which in return depends on its application time34.Figure 4Iodine value (meq kg−1) of black cumin as affected by moringa leaf extract applied at various growth stages.Full size imageTotal free amino acidsThe data presented in Table 2 revealed that moringa leaf extract concentrations and application stages had significantly affected total free amino acid content of black cumin crop during rabi 2019-20 under agro-climatic conditions of Haripur whereas, their interaction remained non-significant. The planned mean comparison of control vs rest and water spray vs rest had significant effect on total free amino acids of black cumin. Highest amino acids (336.3) were observed with the application of moringa leaf extract @ 20%, followed by application of moringa leaf extract @ 10%. Regarding application stages, highest total free amino acids (364.2) were observed with the application of moringa leaf extract at 40 + 80 + 120 days after sowing, followed by (355.9) application of MLE at 40 + 80 days after sowing. Lowest total free amino acids (290.3) were recorded with moringa leaf extract sprayed at 40 days after sowing. Several investigations have demonstrated that MLE can alter both primary and secondary metabolism, resulting in an increase in antioxidant molecule concentrations35,36. The content of phenolic antioxidants, total soluble proteins, and total free amino acids increased in spinach plants treated with synthetic growth regulators and MLE26. MLE can also increase fruit quality metrics in ‘Kinnow’ mandarins, such as soluble solid contents, vitamin C, sugars, total antioxidant, phenolic contents, and superoxide dismutase and catalase enzyme activities, when treated at various growth stages37.Total phenolicPhenolic have acquired much importance because of their properties of disease preventing and health promoting. The effect of moringa leaf extract concentrations, stage of application and their interaction is presented in Table 2. Analysis of variance revealed that moringa leaf extract concentrations and stage of application of moringa leaf extract had significant effect on total phenolic content of black cumin while their interaction remained non-significant. Our results depict that all MLE levels enhanced the total phenolic content of black cumin leaves relative to the control. Highest phenolic content (71.59 mg g−1) was observed with application of moringa leaf extract at the rate of 20%, followed by (68.72 mg g−1) moringa leaf extract application at the rate of 10%. Regarding application stages, highest phenolic content (81.23 mg g−1) was observed with the application of moringa leaf extract at growth stage-7 (40 + 80 + 120 days after sowing), followed by (76.66 mg g−1) stage-6 (80 + 120 days after sowing), whereas, lowest phenolic content (55.25 mg g−1) was observed in crop sprayed with moringa leaf extract at stage-3 (120 days after sowing). In the medicinal, biological, and agricultural areas, phenolic and their derivatives gained scientists attention. Recent studies had focused on their potential as antioxidant-rich natural chemicals38. The increased content of phenolics, flavonoids, and phytohormones in moringa leaves, which may have contributed to the enhanced total phenolic content in black cumin leaves, can be linked to the higher content of phenolics, flavonoids, and phytohormones in MLE treated plants26. Furthermore, the proper concentrations of minerals, vitamins, and -carotene found in moringa leaves may have influenced metabolic processes in a way that increased the internal phenolic content in black cumin leaves, either directly or indirectly39. Therefore, these aspects assist MLE to serve as growth enhancer and natural antioxidant40. Our results supported by the previous report of Nasir et al.37 who revealed that the total phenolic content was enhanced as a result of MLE application at critical stages of plant growth. More

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