Nauki Biologiczne i Rolnicze

Journal of Plant Protection Research

Zawartość

Journal of Plant Protection Research | 2021 | vol. 61 | No 2

Abstrakt

The high sensitivity of beans to herbicides is one of the limiting factors regarding the management of dicot weeds in bean crops. Protoporphyrinogen oxidase (PPO) inhibition is an important mechanism of action that has unregistered molecules with potential use in bean crops. The objectives of this study were to investigate the tolerance of Brazilian bean cultivars to distinct PPO inhibitors and to determine the existence of cross-tolerance in cultivars to the different PPO inhibitor chemical groups. In the first and second experiments, the BRSMG Talismã, Jalo Precoce, BRS Esplendor, and IPR 81 cultivars were subjected to saflufenacil doses pre- (0, 9.6, 14.1, 20.5, 30.0, and 43.8 g a.i. ‧ ha–1) and post-emergence (0, 0.7, 1.0, 1.5, 2.1, and 3.1 g a.i. ‧ ha–1). In the third experiment, the tolerance of 28 bean genotypes to saflufenacil (20.5 g a.i. ‧ ha–1) in pre-emergence was determined. In the fourth, fifth, sixth and seventh experiments, we investigated the cross-tolerance of bean to the fomesafen, flumioxazin, sulfentrazone, and saflufenacil herbicides, respectively. Even very low saflufenacil doses in post-emergence caused plants of all cultivars to die rapidly; therefore, the tolerance was much lower at this application time than in pre-emergence. There was high tolerance variability to saflufenacil among the 28 cultivars. The bean tolerance to fomesafen, flumioxazin, sulfentrazone, and saflufenacil applied pre-emergence depended on the cultivar and dose. Fomesafen was highlighted owing to its higher selectivity in relation to the different cultivars. No cross-tolerance pattern to the PPO inhibitor chemical groups applied in pre-emergence was observed among the evaluated bean cultivars. The results of this study could be of significance to farmers and technical assistance personnel, as well as for future research on cultivar breeding and the elucidation of biochemical and genetic mechanisms involved in herbicide tolerance.
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Autorzy i Afiliacje

Antonio Pedro Brusamarello
1
Michelangelo Muzell Trezzi
1
Fortunato de Bortoli Pagnoncelli Júnior
1
Paulo Henrique de Oliveira
1
Taciane Finatto
1
Marcos Vinícius Jaeger Barancelli
1
Bruno Alcides Hammes Schmalz
1
Patrícia Bortolanza Pereira
1

  1. Department of Agronomy, Federal Technological University of Paraná (UTFPR), Brazil

Abstrakt

The olive psyllid Euphyllura straminea Loginova (Hemiptera: Aphalaridae) is one of the most important pests of olive trees in Iran. To determine this pest’s economic injury level (EIL) and to evaluate the relationship between density of nymphs (DON) and yield loss, different densities of olive psyllid nymphs were maintained on olive trees by different insecticide concentrations. Counting nymphs on olive terminal shoots was done to determine nymph abundance at the end of nymphal stage. Different densities of olive psyllid nymphs resulted in significantly different yield losses of olive trees. Regression analysis was used to determine the relationship between nymph density and yield loss. Considering pest management costs, the market value of olive, and insecticide efficiency, economic injury levels were evaluated from 4.08 to 7.14 nymphal days. The olive psyllid EIL values could be used to plan a pest control program in Zanjan and Guilan provinces.
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Autorzy i Afiliacje

Aref Marouf
1
Mohammadreza Abbasi Mojdehi
2
Shamsollah Najafi
1

  1. Plant Protection Department, Agricultural and Natural Resources Research Center of Zanjan Province (AREEO), Iran
  2. Plant Protection Department, Agricultural and Natural Resources Research Center of Guilan Province, Rasht, Iran

Abstrakt

The miner fly Liriomyza sativae (Blanchard) (Diptera: Agromyzidae) is an insect of economic importance for tomato culture. The conventional control with insecticides is complex due to the mining eating habit that provides protection to the larvae inside the leaves. Therefore, farmers can opt for biological control agents, or substances that provide protection to the plant. Thus, the objective of our research was to evaluate the use of silicon to induce resistance in tomato plants against L. sativae. The results showed that in tomato plants treated with SiO2/F and K2SiO3/F there was a reduction in the net reproduction rate (Ro), in the intrinsic rate of increase in number (rm), in the finite rate of increase (λ), in the average interval between generations (IMG), in the doubling time (TD), in the number of eggs/ female/day and the accumulated egg laying of F1 females of L. sativae. The products SiO2/F and K2SiO3/F gave the tomato a protective effect against injuries caused by L. sativae.
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Autorzy i Afiliacje

Adamastor Pereira Barros
1
ORCID: ORCID
Hugo Bolsoni Zago
2
ORCID: ORCID
Dirceu Pratissoli
2
ORCID: ORCID
Paulo Cezar Cavatte
3
ORCID: ORCID
Julielson Oliveira Ataide
2
ORCID: ORCID

  1. Entomology Department, Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, Brazil
  2. Entomology Department, Universidade Federal do Espírito Santo, Campus Alegre, Alegre, Brazil
  3. Biology Department, Universidade Federal do Espírito Santo, Campus Alegre, Alegre, Brazil

Abstrakt

Several national rice centers in Indonesia have used acetolactate synthase herbicide inhibitors for years, especially in several regions of Lampung and West Java provinces. This practice has led to the failure of the application of bensulfuron-methyl herbicide to control Monochoria vaginalis (Burm. f.) C. Presl. The purposes of this study were to confirm that the failure of herbicide application in several areas of the provinces of Lampung and West Java was caused by weed resistance, and to determine the level of resistance. A resistance test of M. vaginalis was performed using the whole plant pot test method and split plot design with three replications. Monochoria vaginalis which indicated resistance was sampled from several regions, namely Sapto Mulyo, Ramadewa, Sarijaya, and Kalentambo. The susceptible samples of M. vaginalis as a control were taken from Cibodas and Sumberagung. The six levels of doses of herbicide bensulfuron-methyl used were: 0, 80, 160, 320, 640 and 1,280 g ha–1. The experimental results show that M. vaginalis from Sapto Mulyo, Ramadewa, Kalentambo and Sarijaya was confirmed to have developed into weeds resistant to bensulfuron-methyl herbicide. Monochoria vaginalis from Saptomulyo, Kalentambo and Sarijaya were included in the high resistance category with a resistance ratio of more than 12, while M. vaginalis from Ramadewa was included in the moderate resistance category with a resistance ratio of 9.39.
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Autorzy i Afiliacje

Denny Kurniadie
1
ORCID: ORCID
Ryan Widianto
2
Dedi Widayat
1
Uum Umiyati
1
Ceppy Nasahi
3

  1. Department of Agronomy, Faculty of Agriculture, Universitas Padjadjaran, Bandung, Indonesia
  2. Graduate student, Faculty of Agriculture, Universitas Padjadjaran, Bandung, Indonesia
  3. Department of Pest and Diseases, Faculty of Agriculture, Universitas Padjadjaran, Bandung, Indonesia

Abstrakt

Annual losses of cocoa in Ghana to insect pests are significant. The use of integrated pest management (IPM) tools is critical for effective pest management. Previous studies on the subject have considered how farmers perceive the economic impact of insect pests on cocoa. These studies however did not investigate farmers’ ability to identify pests, associated damage symptoms and their implications for pest management. The current study, therefore, assessed farmers’ ability to correctly associate insect damage with the pest species that caused it. A total of 600 farmers were interviewed in the Eastern, Ashanti, Western, Brong Ahafo and Central Regions of Ghana with a structured open and closedended questionnaire. Most farmers (>85%) were unable to correctly identify and associate pests to their damage. The majority (>80%) of farmers also could not link the immature stages of insect pests to their adult stages. Wrong identification of the major pests (>85%) led to a wide variation in the timing of insecticide application amongst farmers. The majority of the farmers (60%) interviewed had not received training in insect pest identification. The study shows that 90% of the farmers, who had received some training, got it from the Cocoa Health and Extension Division (CHED) of the Ghana Cocoa Board (COCOBOD). Almost all respondents (98%) agreed that correct pest identification is critical for effective pest control. The importance of pest identification and monitoring as a component of IPM is discussed.
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Autorzy i Afiliacje

Godfred Kweku Awudzi
1
Richard Adu-Acheampong
1
Silas Wintuma Avicor
1
Yahaya Bukari
2
Millicent Adomaa Yeboah
3
Edmond Kwadwo Oti Boateng
4
Stephen Kwame Ahadzi
1

  1. Entomology, Cocoa Research Institute of Ghana (CRIG), Ghana
  2. Plant Pathology, Cocoa Research Institute of Ghana (CRIG), Ghana
  3. Extension, Cocoa Health and Extension Division (CHED), Ghana
  4. Social Science and Statistics Unit (SSU), Cocoa Research Institute of Ghana (CRIG), Ghana

Abstrakt

Presently, finding effective, simple, inexpensive, hygienic and safe pest control agents are the biggest challenges in management of stored product insects, where those features are available in most physical factors. The insecticidal efficiency of four diversified physical control agents (ultraviolet and microwave irradiations, thermal remediation and silica nanoparticles) were assayed against the most common coleopteran insect species ( Sitophilus oryzae L. and Tribolium castaneum Herbst) on stored wheat. Exposing tested insects to microwave irradiations (2450 MHz) for 25 sec gave preventive efficiency for stored material, which reached 97.68 and 99.02%, respectively. Sufficient exposure periods to kill 50% of the coleopteran adults (LT50%) were 13 and 14 sec, respectively. For effective control with UV radiations, S. oryzae should be exposed for 12 h and T. castaneum for 24 h. An exposure period of 24 h caused progeny reduction 95.24 and 89.72% and gave preventive efficiency of 94.25 and 93.37%, respectively. Values of LT50% were 56.76 and 74.04 h, respectively. Exposing infested samples of the tested species to 70oC for 10 min killed 100% of adults and caused complete cessation of egg laying. Furthermore, 65°C or 70°C caused full progeny reduction. The lowest level of stored product weight loss (1.15 and 1.35%, respectively) occurred at 70°C, where sufficient exposure temperatures to kill 50% of the coleopteran adults (LTD50%) were 60.95°C and 61.63°C, respectively. Synthetic silica nanoparticles (SSiNPs) were more toxic against the tested populations than bio-silica nanoparticles (BSiNPs) after 48–72 h. A concentration of 1.00 g kg–1 of tested silica nanoparticles caused significant reduction in adult populations, saved wheat grain vitality and gave least lost weights of flour (3.35–6.85%).
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Autorzy i Afiliacje

Khalil A. Draz
1
Magdy I. Mohamed
2
Reda M. Tabikha
1
Adnan A. Darwish
1
Mohamed A. Abo-Bakr
1

  1. Plant Protection Department, Faculty of Agriculture, Damanhour University, Damanhour, Egypt
  2. Stored Product Pests Department, Plant Protection Research Institute, Agricultural Research Center, Alexandria, Egypt

Abstrakt

Currently, Prosopis laevigata (mesquite) has been affected by the Bruchinae coleoptera pest, whic feeds on its seed and causes significant losses in production and grain storage. In the Hñähñu community El Alberto (Ixmiquilpan, Hidalgo, Mexico), the use of aqueous extracts from garlic and nettle as botanical insecticides against different pests in agricultural fields is a known practice. Herein, we assess the efficacy of the method known by locals in the protection of mesquite seeds. Two tests were conducted: 1) Insecticidal effect on adult bruchins, and 2) Seed preservation test from Bruchinae infestation, with a germination test in seeds exposed to the treatments. There are probable insecticidal effects on immature stages of Bruchinae since there were no mortality effects on their adults during the first test. Mortality on adults in the second test was 75.6% with garlic and 50% with nettle. Nettle extract had more efficacy in seed protection with an infestation rate of 4%, whereas 27.5% of the seeds exposed to garlic extract were infested. Seed germination rate was 2.38% with nettle extract, and 1.19% with garlic extract. The method known by local inhabitants requires modifications to increase its efficacy and possible use in Integrated Pest Management in the future.
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Autorzy i Afiliacje

Mariana González-Macedo
1
Nathalie Cabirol
1
Marcelo Rojas-Oropeza
1

  1. Functional Soil Microbial Ecology and Environmental Protection Group − Department of Ecology and Natural Resources, Facultad de Ciencias − Universidad Nacional Autonoma de Mexico, Mexico

Abstrakt

The study of herbicide dynamics in the soil and their interaction with the components of the environment makes it possible to ensure the selectivity of crops and the agronomical efficiency. The aim of this research was to evaluate the influence of soil physicochemical properties on the emergence and growth of soybean, with pre-emergence application of various saflufenacil rates. An experiment was carried out in a greenhouse with a completely randomized design, testing different soil types containing “Erechim”, “Santa Maria” and “Eldorado do Sul”, at different saflufenacil rates: 0, 12.5, 25, 50, 100, 200, 400 g a.i. · ha–1. The application was performed 1 day after soybean sowing, and analyzed variables were: the phytotoxicity emergence of seedlings, dry mass and height of the soybean. The saflufenacil effective dose of 50% response in soybean (ED50) and the characteristics of the soils showed that the soil contained clay and sand which were the components most related to the saflufenacil availability to the plants. A lower ED50 by phytotoxicity to the soybean was found in soil with lower and greater content of clay and sand, respectively. The physicochemical properties of soil influenced the saflufenacil activity, having greater potential of injury to soybean in the soil from Eldorado do Sul, due to its clay and sand content.
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Autorzy i Afiliacje

Geovana Facco Barbieri
1
Cassiano Salin Pigatto
1
Glauco Pacheco Leães
2
Nelson Diehl Kruse
2
Dirceu Agostinetto
1
André da Rosa Ulguim
2

  1. Plant Protection Department, Federal University of Pelotas, Av. Eliseu Maciel, 96160-000, Capão do Leão, Rio Grande do Sul, Brazil
  2. Plant Protection Department, Federal University of Santa Maria, Av. Roraima, 1000, 97105-900, Santa Maria, Rio Grande do Sul, Brazil

Abstrakt

Plants can recognize molecules derived from pathogens and trigger systemic acquired resistance (SAR). In phytopathogenic bacteria, elicitors are constituent components of cellular structures, such as flagellin. We sought to select structural components of Xanthomonas spp. incompatible with tomato, aiming to control bacterial spot ( Xanthomonas perforans). Initially, cell suspensions from 11 Xanthomonas spp. isolates were infiltrated into the leaves to assess their ability to cause a hypersensitivity response (HR) and the incompatible ones had their flagellin purified. The flagellin of the isolates were first applied at different concentrations, via infiltration and spraying. The pathogen, X. perforans, was inoculated after 24 h, to assess whether there would be any harmful reaction. No harmful reaction was observed in any treatment. Then, a second experiment was conducted to assess the severity of all isolates, at a concentration of 8.35 μg · ml–1, via spraying, infiltration, and soil. The greatest reduction in Area Under the Disease Progress Curve (AUDPC) was observed in the treatment with XapRR, applied via spraying. Thus, prospecting for elicitors is the first step in developing a product for agricultural use. The flagellin elicitor of XapRR is promising and capable of producing these molecules on a large scale.
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Autorzy i Afiliacje

Camila Tonelotti Simões
1
ORCID: ORCID
Valdeir Nunes Carvalho
1
ORCID: ORCID
Bernardo de Almeida Halfeld-Vieira
2
ORCID: ORCID

  1. Faculdade de Ciências Agronômicas, Universidade Estadual Paulita “Júlio de Mesquita Filho”, Botucatu, Brazil
  2. Fitopatologia, Embrapa Meio Ambiente, Jaguariúna, São Paulo, Brazil

Abstrakt

The ability of parasitoids in locating hosts determines their success in suppressing the pest population. Chemical stimuli emitted from food products and hosts provoke the searching behavior of parasitoids. Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae) is a generalist idiobiont ectoparasitoid of coleopteran pests in stored products. In the current study, the behavioral responses of A. calandrae females were evaluated regarding host food and different life stages of the host, Callosobruchus maculatus F. (Coleoptera: Chrysomelidae), using a Y-tube olfactometer. The parasitoid was offered uninfested chickpea kernels, damaged chickpea without larvae of C. maculatus, damaged chickpea with preferred stage (4th instar) larvae of C. maculatus, uninfested chickpea + C. maculatus adults, and eggs of C. maculatus on chickpea. In another test, the preference of A. calandrae for either damaged chickpea without larva of C. maculatus or damaged chickpea with nonpreferred stage (1st instar) larvae of C. maculatus was studied. The results showed that the females did not prefer uninfested chickpea kernels and adults of C. maculatus. However, they were attracted to damaged kernels with or without larvae, and the kernels containing eggs of C. maculatus. When the female parasitoids had a choice between damaged chickpea without larva of C. maculatus and damaged chickpea with 1st instar larva, they did not prefer one over the other. The results of this investigation can be helpful for using A. calandrae as a biological control agent in stored products.
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Autorzy i Afiliacje

Masoomeh Moosavi
1
Nooshin Zandi-Sohani
1
Ali Rajabpour
1

  1. Plant Protection Department, Agricultural Sciences and Natural Resources University of Khuzestan, Ahvaz, Iran

Abstrakt

In 2016, bacterial canker symptoms, often with dried ooze, were observed on Salix alba plants in municipal lands and parks in Kerman and Fars provinces, Iran. To determine the causative agent, samples were collected from symptomatic trees, and macerates of the affected bark tissues were plated on sucrose nutrient agar (SNA). Ten isolates were identified by phenotypic characterization, pathogenicity tests, and two of them further confirmed identity using sequence analysis of the partial of 16S rRNA and gyrB genes, and phylogenetic analysis. The isolates showed the highest identity (99–100%) with Brenneria salicis. To our knowledge, this is the first report of watermark disease on S. alba caused by B. salicis in Iran.
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Autorzy i Afiliacje

Esmaeil Basavand
1
ORCID: ORCID
Pejman Khodaygan
1
Mojtaba Dehghan-Niri
2
Saman Firouzianbandpey
1

  1. Department of Plant Pathology, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran
  2. Department of Plant Pathology, Ferdowsi University of Mashhad, Mashhad, Iran

Abstrakt

The tea plant [ Camellia sinensis (L.) O. Kuntze] is one of the most significant commercial crops in Iran. Symptomatic leaves including chlorina on the edge of the leaf, and multiple necrotic ring blotches on mature leaves from different tea gardens were observed in northern Iran. RT-PCR analysis and transmission electron microscopy observations were applied to characterize the causal agent of tea leaf discoloration. Sequence analyses of the fragments revealed that all the samples were infected with tea plant necrotic ring blotch virus (TPNRBV). To our knowledge this is the first report of TPNRBV in Iran and the second in the world.
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Autorzy i Afiliacje

Eisa Nazerian
1
ORCID: ORCID
Hossein Bayat
2

  1. Department of Technology and Production Management, Tea Research Center, Horticultural Sciences Research Institute, Lahijan, Iran
  2. Department of Technology and Production Management Ornamental Plant Research Center, Horticultural Sciences Research Institute, Mahallat, Iran

Instrukcja dla autorów

Instructions for Authors

Manuscripts published in JPPR are free of charge. Only colour figures and photos are payed 61.5 € per one colour page JPPR publishes original research papers, short communications, critical reviews, and book reviews covering all areas of modern plant protection. Subjects include phytopathological virology, bacteriology, mycology and applied nematology and entomology as well as topics on protecting crop plants and stocks of crop products against diseases, viruses, weeds, etc. Submitted manuscripts should provide new facts or confirmatory data. All manuscripts should be written in high-quality English. Non-English native authors should seek appropriate help from English-writing professionals before submission. The manuscript should be submitted only via the JPPR Editorial System (http://www.editorialsystem.com/jppr). The authors must also remember to upload a scan of a completed License to Publish (point 4 and a handwritten signature are of particular importance). ALP form is available at the Editorial System. The day the manuscript reaches the editors for the first time is given upon publication as the date ‘received’ and the day the version, corrected by the authors is accepted by the reviewers, is given as the date ‘revised’. All papers are available free of charge at the Journal’s webpage (www.plantprotection.pl). However, colour figures and photos cost 61.5 € per one colour page.

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All text should be written in a concise and integrated way, by focusing on major points, findings, breakthrough or discoveries, and their broad significance. All running text should be in Times New Roman 12, 1.5 spacing with all margins 2.5 cm on all sides.

Original article

The original research articles should contain the following sections: Title – the title should be unambiguous, understandable to specialists in other fields, and must reflect the contents of the paper. No abbreviations may be used in the title. Name(s) of author(s) with affiliations footnoted added only to the system, not visible in the manuscript (Double Blind Reviews). The names of the authors should be given in the following order: first name, second name initial, surname. Affiliations should contain: name of institution, faculty, department, street, city with zip code, and country. Abstract – information given in the title does not need to be repeated in the abstract. The abstract should be no longer than 300 words. It must contain the aim of the study, methods, results and conclusions. If used, abbreviations should be limited and must be explained when first used. Keywords – a maximum of 6, should cover the most specific terms found in the paper. They should describe the subject and results and must differ from words used in the title. Introduction – a brief review of relevant research (with references to the most important and recent publications) should lead to the clear formulation of the working hypothesis and aim of the study. It is recommended to indicate what is novel and important in the study. Materials and Methods – in this section the description of experimental procedures should be sufficient to allow replication. Organisms must be identified by scientific name, including authors. The International System of Units (SI) and their abbreviations should be used. Methods of statistical processing, including the software used, should also be listed in this section. Results – should be presented clearly and concisely without deducting and theori sing. Graphs should be preferred over tables to express quantitative data. Discussion – should contain an interpretation of the results ( without unnecessary repetition) and explain the influence of experimental factors or methods. It should describe how the results and their interpretation relate to the scientific hypothesis and/or aim of the study. The discussion should take into account the current state of knowledge and up-to-date literature. It should highlight the significance and novelty of the paper. It may also point to the next steps that will lead to a better understanding of the matters in question. Acknowledgements – of people, grants, funds, etc. should be placed in a separate section before the reference list. The names of funding organizations should be written in full. References In the text, papers with more than two authors should be cited by the last name of the first author, followed by et al. (et al. in italics), a space, and the year of publication (example: Smith et al. 2012). If the cited manuscript has two authors, the citation should include both last names, a space, and the publication year (example: Marconi and Johnston 2006). In the Reference section, a maximum of ten authors of the cited paper may be given. All references cited in the text must be listed in the Reference section alphabetically by the last names of the author(s) and then chronologically. The year of publication follows the authors’ names. All titles of the cited articles should be given in English. Please limit the citation of papers published in languages other than English. If necessary translate the title into English and provide information concerning the original language in brackets (e.g. in Spanish). The list of references should only include works from the last ten years that have had the greatest impact on the subject. Older references can be cited only if they are important for manuscript content. The full name of periodicals should be given. If possible, the DOI number should be added at the end of each reference. The following system for arranging references should be used: Journal articles Jorjani M., Heydari A., Zamanizadeh H.R., Rezaee S., Naraghi L., Zamzami P. 2012. Controlling sugar beet mortality disease by application of new bioformulations. Journal of Plant Protection Research 52 (3): 303-307. DOI: https://doi.org/10.2478/v10045-012-0049-9 Online articles Turner E., Jacobson D.J., Taylor J.W. 2011. Genetic architecture of a reinforced, postmating, reproductive isolation barrier between Neurospora species indicates evolution via natural selection. PLoS Genetics 7 (8): e1002204. DOI: https://doi.org/10.1371/journal.pgen.1002204 Books Bancrof J.D., Stevens A. 1996. Theory and Practice of Histological Techniques. 4th ed. Churchill Livingstone, Edinburgh, UK, 776 pp. Book chapters Pradhan S.K. 2000. Integrated pest management. p. 463-469. In: "IPM System in Agriculture. Cash Crop" (R.K. Upadhyaya, K.G. Mukerji, O.P. Dubey, eds.). Aditya Books Pvt. Ltd. New Delhi, India, 710 pp. Online documents Cartwright J. 2007. Big stars have weather too. IOP Publishing PhysicsWeb. Available on: https://doi.org/10.1371/journal.pgen.1002204

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Rapid communications

Rapid communications should present brief observations which do not warrant the length of a full paper. However, they must present completed studies and follow the same scientific standards as original articles. Rapid communications should contain the following sections: Title Abstract - less than 300 words Key words - maximum 6 Text body Acknowledgements References The length of such submissions is limited to 1500 words for the text, one table, and one figure.

Reviews

Review articles are invited by the editors.Unsolicited reviews are also considered. The length is limited to 5000 words with no limitations on figures and tables and a maximum of 150 references. Mini-Review articles should be dedicated to "hot" topics and limited to 3000 words and a maximum two figures, two tables and 20 references.

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