Comparative Efficiency and Reproducibility of Albit®

Development and implementation of pesticides with living organisms or their metabolic products as active ingredients is a rapidly developed field of agricultural biotechnology at the present moment. The annual increase of bioformulations production in the world is up to 10-15%, whereas for traditional chemical pesticides, the increase equals 1-2% [1]. Bioformulations have a number of benefits in comparison with traditional plant-protecting chemical formulations, among of them are: ecological compatibility, low toxicity level, low prices, versatility of use and wide spectrum of effect. In recent years, in accordance with The list of pesticides and agrochemicals allowed for use on the territory of the Russian Federation, the amount of registered biogenic formulations has increased to approximately 10% of the total amount of registered pesticides. According to the figures from the Economic laboratory of the All-Russia Institute of Plant Protection, consumption of biological formulations increased by 2.7 times compared to 1999, and 26 different formulations were used in total. The biological load consisted of 0.008 kg/ha of agricultural land, whereas in 1995-1999 biological load of bioformulations equaled 0.003 kg/ha [2].

At the same time, at the present moment it is not possible to state with certainty that bioformulations are able to compete with chemically synthesized pesticides as an equal. Among the disadvantages of bioformulations are: their short shelf life, strict requirements for storage and application procedures and low compatibility with standard chemical plant-protecting agents. In terms of application effectiveness, the competitiveness of bioformulations is critically affected by 2 factors, according to many researchers:

1. Effectivity of bioformulations is still inferior to that of chemical formulations. Though in a number of tests bioformulations are as good as or even better than chemical formulations, general figures are not in favor of bioformulations, and that restrains their wider implementation;
2. Even when effective, bioformulations have a second substantial disadvantage — low reproducibility of their effect. The behavior of living microorganisms in biological environment when put on the plants is influenced by a variety of factors (temperature, humidity, state of natural microbial community, the phase of plant development, etc.), so the effect of their use is often low or unpredictable. Even if microbes are often highly efficient against pathogens in the laboratory conditions, there is no guarantee of their high efficiency when transferred to the root area or on the surface of plants, where they get into stressful environmental conditions and have to compete with indigenous microorganisms.

During the development of Albit, very close attention was paid to overcoming the above-mentioned "birth defects" of bioformulations.

Firstly, it was possible to abandon the use of live bacteria in the product — the basis of the formulation was formed by individual active substance of microbial origin (poly-beta-hydroxybutyrate, PHB). As we know from the experience of other pesticide development, this method helps to increase their effectiveness. For example, chemically pure pyrethroids have a higher insecticidal activity than decoction of chamomile (Pyrethrum corymbosum) leaves, from which they were first extracted. On the basis of active substances extracted from microorganisms, two new classes of chemical fungicides were proposed: cyanopyrroles and strobilurines originally discovered in bacteria, Pseudomonas, and fungus Strobilurus (Marasmius) [3], respectively. On this basis, the company Syngenta developed an effective formulation called Maxim, and the company BASF developed formulations named Strobi and Quadris.

Secondly, in development of the Albit formulation, in contrast to its predecessors (Agat-25 and Agat-25k), a pure culture of bacteria was not used for production of poly-hydroxybutyrate, but an association of two bacterial strains (Pseudomonas aureofaciens and Bacillus megaterium). Many examples have previously shown that the use of associations provides higher efficiency of bioformulations than pure cultures [4]. It was also proved that associations provide higher reproducibility of the effect compared to pure bacteria [5]. However, despite these obvious advantages of associations, until recently they were little used in the development of fungicidal bioformulations, and plant-growth regulators. For development of the Albit formulation, a high potential of microbial associations was used, which allowed, in particular, to increase the content of the active substance (PHB) by about 1.5 times.

Experts in agriculture know well, that the result of application of even highly effective pesticides can noticeably vary depending on year, field specificity, agricultural background, phytosanitary conditions, terms of treatment. One of the purposes of Albit development was overcoming of low reproducibility, which is a common defect of most of the biostimulants. Reproducibility can be quantitatively characterized by the ratio of standard deviation of all experiments and the arithmetical mean (variation coefficient CV). According to averaged results of all field trials, CV of Albit is 52%, biological products and plant growth regulators (without Albit) – 130%, synthetic chemical fungicides – 59%. Therefore, the ability of Albit to provide stable yield increase (reciprocal value for CV) was 2 times higher than that of the other analogues and 26% higher than that of chemical standards. Therefore, high reliability and reproducibility is one of the main advantages of Albit (Fig. 1).

Fig. 1. Reproducibility of effect of Albit and other products (according to averaged results of 250 field trials). Yield increase and reproducibility of Albit are taken for 100%. Right - change of average yield increase under influence of Albit and other biostimulants in field trials: in variant with Albit – amplitude of variation is less, reproducibility is higher than in variants with biostimulants

Effectiveness and reproducibility of Albit in comparison with other formulations were quantitatively evaluated in a series of comparative field tests. Field (plot and large scale field) experiments comparing Albit with reference products were carried out in 1997-2004 in the following soil-climatic zones of the Russian Federation: North-West, Central, Central Chernozem, North Caucasus, Volga-Vyatka, Volga, Ural, East Siberia and West Siberia. In general, for comparing Albit and reference samples, registration trials data were used (registration trials are tests where it is mandatory to use reference samples), and also data from similar tests carried out in accordance with generally accepted guidelines for pesticide testing [6]. The area of the test sites for plot tests ranged from 5 to 100 m², for field tests – 1.0–2.5 ha, with 3 to 5 times replications.

As Albit is registered in Russia as a plant-growth regulator and fungicide (including fungicidal seed-pretreatment formulation), it was compared with pesticides having a similar effect: the most commonly used in practice plant-growth regulators, biofungicides, chemical fungicides and seed-pretreatment formulations. In 125 field trials, the protective effect of Albit and reference samples was evaluated, 162 trials of growth-stimulation (yield increase) effects were compared.

Comparative Efficiency of Albit

The following specifications were used as criteria for comparative effectiveness of formulations:

1. as a characteristic for agricultural efficiency (growth-stimulating effect) – a ratio of the yield increase to a control provided by Albit to increase in the case of the reference [7];
2. as a characteristic for biological efficiency against diseases (fungicidal effect), a ratio of the biological control efficacy (BE) of Albit to the BE of the reference was used;
3. as a characteristic for economic efficiency, analogous relationship of net income per hectare (RUR/ha) in cases for Albit and reference was used [8].

The data obtained have been averaged for each formulation separately. The results of all field tests of Albit in comparison to the reference are summarized in Table 1. Taking into consideration scientific ethics, we labeled the formulations with their active substances instead of the commercial brands, and the formulations were assigned notional numbers.

For example, the table indicates that Albit has been compared to fungicide No. 1 in the tests by the All-Russia Institute of Plant Protection, conducted in 2003 and 2004. The tests were conducted with potato. The yield increase upon treatment with Albit on average surpassed the reference in 1.13 times, and net income per hectare (economic efficiency) — in 3.41 times. The fungicide effect of Albit appeared to be weaker than with the reference, i.e. Albit’s biological efficacy against diseases on average accounted for just 0.89 (89%) of the reference.

From the data of the table we conclude that by the effect on plants' yield that Albit was proven to be more effective than 29 of the studied formulations (74%) and 10 references (26%) surpassed Albit. The economic efficiency of Albit surpassed its peers in the great majority of cases (96%).

Summarizing the data for the two groups of formulations shown in the table, it was found that Albit on average is 2.24 times better in terms of yield compared to peer bioformulations and underperforms chemical fungicides by a factor of just 1.06 times. The biological efficacy of Albit was 76% of the chemical fungicides’ effect and 160% of other bioformulations and growth stimulators. To conclude, the economic efficiency of Albit surpassed both chemical (by 1.89 times) and biological (by 2.24 times) peers. Despite certain approximation of the examined averages, it nevertheless presents the general idea of Albit’s placement among other plant-protecting formulations.

Comparative reproducibility of the effect of Albit

When characterizing virtually every pesticide, one may showcase specific successful tests, where the formulation performance compared to controls and references was high. Such tests you may often find in especially in marketing brochures of their manufacturers. More representative is an aggregated approach, when test series under different conditions are utilized, and on these grounds a general vision of a substance’s average efficiency is constructed. Such an attempt to examine the average efficiency of Albit was taken in the previous chapter.

But apart from the average characteristics of efficiency, reproducibility of effects from formulations is also of great importance. Stability and reproducibility of the effect in various soil, agrochemical, agro-climatic and phytosanitary conditions is an important issue especially for formulations of biological origin. Many of them, specifically those produced from live microorganisms, enjoying relatively high average efficiency, are subject to high degree of variation in the results in real circumstances depending on specific agrochemical and agro-climatic conditions. With reference to published data, we know that the effect from treatment with bioformulations may vary greatly – from yield increase by 40% to its reduction by 20% depending on the conditions of treatment [9]. The main cause for low reproducibility of microorganisms’ activity is the great influence of environmental conditions on their physiological processes and high probability of elimination of introduced populations not able to compete with natural microorganisms of the phyllosphere and rhizosphere [10].

The vast accumulation of comparative data for Albit and references from field tests allows a comparison of the reproducibility of its effect (Table 2). Here the same indicators as for average efficiency were used: yield increase to control (in %) and biological efficacy against diseases (in %).

For a quantitative estimation of field tests sample variation in natural science research, variance is more often used [11], according to the formula:

, where

s² – variance,
n – number of observations (trials),
x_i – result of an i observation,
x_ñð – arithmetic mean of all observations.

Standard deviation s is also widely used, that is a positive square root of the variance:

Standard deviation is a main estimator for variation of characteristics. This parameter is not dependant on the number of observations, and that is why it may be used for comparative estimation of the variation of homogeneous characteristics. However, the average mean square is not widely used as an estimator for variation of characteristics because the output is a denominate number. So, for the most appropriate estimation of variation of characteristics without respect to units of measurement, it is a common practice to express as a percentage of an arithmetic average [12]. The output of such transformation is called coefficient of variation (designated as CV or V) and is calculated as:

, where

V – coefficient of variation, %,
s – standard deviation,
x_ñð – arithmetic mean of all trials.

Coefficient of variation V is a characteristic for let say ‘hitting a target’, the ability of a formulation to demonstrate stable average efficiency from one test to another, not deviating significantly under different factors (Fig. 2). The higher the V ratio, the more significant the dispersion of treatment results with a specific formulation, and the lower the reproducibility of its efficiency. The coefficient of variation is an inverse parameter for reproducibility: the higher the ratio, the farther are the outputs of field tests from the average, resulting in a lower reproducibility.

Fig. 2. Formulations with high (à) and low (b) reproducibility of action. Big circle – average efficiency, small circle – results of single experiments

Examination of the growth-stimulating and protective effects of Albit and reference formulations was conducted in field tests (Table 2). To compare the coefficient of variation V for each pair of a reference formulation (VR) and Albit (VA) the ratio (VR / VA) was used. Formulations with a value of VR/ VÀ above 1 are more variable (with lower reproducibility of the effect) compared to Albit. While examining variation in order to receive statistically representative data the formulations with at least 3 comparative tests with Albit were taken into account. Reproducibility of an economic effect was not estimated due to the fact that the insignificant number of observations compromised statistical accuracy.

The majority of studied formulations demonstrated higher dispersion of values and, consequently, lower reproducibility compared to Albit (VR/VÀ > 1). Taking the reproducibility of growth-stimulating effect, Albit surpassed 14 reference formulations (74% of the total) and 5 formulations performed better than Albit (26%). Just some chemical formulations and bioformulations, No. 10 and No. 12, showed lower variability in yield increase than Albit. The reproducibility for the formulations created from individual chemical substances (No. 10, No. 27, Albit, chemical fungicides) was generally higher compared to formulations with live microorganisms as active substances (Nos. 13, 14, 22).

The same situation was encountered for the reproducibility of the fungicide effect from formulations. Even bioformulations with high average efficiency and with official statuses of fungicides (formulations on the basis oå Pseudomonas aureofaciens, Ps. fluorescens, Bacillus subtilis), have demonstrated 1.2-6.5 times lower reproducibility of the fungicide effect compared to growth regulators on the basis of metabolites.

For the final estimation of the reproducibility of effect for the pesticides, we may average the values of their coefficients of variation V (Table 3). Aggregated values of variation ratios clearly indicate already noted tendencies and provide their quantitative estimation for different groups of formulations. The highest value for V was recorded for bioformulations and growth-stimulators (average 130% for yield), but strictly for bioformulations (with microorganisms as active substances) the value is even higher – 225%. Accounting for fungicide effect, generally lower variability across all types of formulations was noticed. Variability for Albit is behind those for bioformulations and approximately equals those of chemical synthesis formulations.

The results obtained can be obviously interpreted in the following way: yield increase thanks to Albit may vary by 0.5 times from year to year, for other bioformulations the variation is 1.3 times, for chemical fungicides – 0.6 times on average. For example, if a farm has received a yield increase of 5 centners of wheat on a hectare thanks to Albit, the next year the yield is expected between 2.5 and 7.5 c/ha. The same holds for the treatment with a ‘statistically average’ pesticide from a group of bioformulations and growth stimulators: if the yield increase is 5 centners/ha, the next year it's going to be between an increase of 11.5 centners/ha and a decrease by 1.5 centners/ha compared to the control.

For the purpose of comparative characteristic of variability of Albit and other formulations, we considered it advisable to average the values by VR / VÀ (Table 2). In the same way, for the purpose of comparative characteristic of the average efficiency, we averaged the ratios Albit/Reference (Table 1). The final outputs are represented in Table 4.

In this way, it is possible to conclude with a high probability (99%) that Albit surpasses peer bioformulations in fungicide effect (their mean fungicide effect is just 62% of that for Albit) and is less effective than chemical fungicides (131% of Albit efficiency). Albit is able to provide approximately the same yield increase compared to chemical formulations (variation is not statistically significant). But in growth-stimulating effect, Albit greatly surpassed its peers (bioformulations demonstrated an average of 45% of increase of Albit).

In economic efficiency, Albit surpasses both chemical and biological formulations in the field tests (P = 99%).

The vast field test material acquired proves that the development of Albit with the use of brand new approaches enabled a result of reproducibility both for growth-stimulating and protective effects two times higher compared to peer bioformulations and growth regulators (Table 4). In this way, one of the major drawbacks of bioformulations has been overcomed – the reproducibility of Albit’s effect has reached the level of chemical fungicides.

References and notes:

1. Menn J.J. biostimulants: has their time come? / J.J. Menn // Journal of Environmental Science and Health, Part B — pesticides, food contaminants, and agricultural wastes. — 1996. — Vol. 31. — P. 383-389.
2. Slobodyanyuk V.M. Application of pesticides: somewhat of statistic / Slobodyanyuk V.M., Kricina V.I. // Plant protection and quarantine. — 2004. — ¹ 7. — p. 13-14.
3. Tyuterev S.L. Treatment of cereal crops seeds / Tyuterev S.L. // Addition to the journal «Plant protection and quarantine», – 2005. – ¹ 3. – P. 89(1)-132(44).
4. Ribalskiy N.G. Biotechnological potential of microorganism consortium / Ribalskiy N.G., Lyah S.P. – Moscow, All-Russia Institute of Patent Information. – Vol. 2. – 1990. – 175 p.
5. Guetsky R. Combining biocontrol agents to reduce the variability of biological control / Guetsky R., Shtienberg D., Elad Y., Dinoor A.// Phytopathology. – 2001. – Vol. 91. – P. 621-627.
6. Methodological instructive regulations for state fungicides, antibiotics and seed-treats testing for agricultural crops / Moscow, Agroproisdat. – 1985. – 281 p.
7. Increase in yield was shown in percent to control. Application of percent indexes allows to compare action of pesticides for different crops with different base yield levels (c/ha), and also to compare effect of pesticides in various agrochemical and climatic regions in different years.
8. In case the of data about net income per hectare were lack in field trial reports, the index of profitability (%) was used to assess economic efficiency.
9. Khojevin P.A. On the way to the theory of microbe fertilizers application / Khojevin P.A., Khorchmaru S.S. // Vestnik of Moscow State University. Ser. Soil Science. — 1995. — ¹ 5. — P. 52-62.
10. Polyanskaya L.M. Microbial Succession in Soil / Polyanskaya L.M., Zvyagintsev D.G. // Physiology and General Biology Reviews. – 1995. – vol. 9. – 68 p.
11. Atlas R.M. Microbial ecology: fundamentals and applications. 3rd ed. Benjamin / Atlas R.M., Bartha R. – Cummings Publishing Co., Redwood City. – 1993. – 504 p.
12. Dospehov B.A. The methodic of field experiment / Dospekhov B.A. – Moscow, Kolos. – 1973. – 331 p.

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