Determinación de la flora micológica del maíz seco y su harina en la parroquía San Juan-cantón Gualaceo

El presente trabajo de tesis tuvo como finalidad evaluar la flora micológica del maíz seco y su harina producidos en la parroquia San Juan - Cantón Gualaceo con el fin de aportar con información sobre la calidad de estos alimentos. Además se evaluaron los siguientes factores para el desarrollo de la flora micológica: temperatura, actividad acuosa, almacenamiento; así como también el crecimiento en distintos medios de cultivos y el uso de la desinfección del grano como parte de la técnica de siembra. La detección, recuento y aislamiento de hongos se realizó de acuerdo a la técnica de recuento en placa por siembra en profundidad para la harina y en el caso del grano por la técnica de siembra directa, con y sin desinfección de la superficie del grano utilizándose el agar MEA (Agar Extracto de Malta), PDA (Agar Papa Dextrosa) y DRBC (Agar Rosa de Bengala Diclorán) y sometiendo a dos tratamientos térmicos (18°C vs 25°C). Se encontró que la micoflora presente en las muestras analizadas corresponde a los géneros Penicillium, Fusarium, Rhizopus, Aspergillus y levaduras, siendo el género predominante Penicillium tanto en el grano como en la harina. La temperatura óptima para el crecimiento fue 25°C; y a su vez el crecimiento fue mejor sin desinfectar las superficies del grano. El crecimiento micológico varió dependiendo del medio, siendo PDA el más óptimo para Penicillium y Rhizopus, mientras que para Fusarium y Aspergillus no hubo diferencia. Igualmente se encontró que la actividad acuosa influyó en forma proporcional al crecimiento micológico y que el almacenamiento controlado influyó positivamente en su disminución.

Colonization of Onions by Endophytic Fungi and Their Impacts on the Biology of Thrips tabaci

Endophytic fungi, which live within host plant tissues without causing any visible symptom of infection, are important mutualists that mediate plant-herbivore interactions. Thrips tabaci (Lindeman) is one of the key pests of onion, Allium cepa L., an economically important agricultural crop cultivated worldwide. However, information on endophyte colonization of onions, and their impacts on the biology of thrips feeding on them, is lacking. We tested the colonization of onion plants by selected fungal endophyte isolates using two inoculation methods. The effects of inoculated endophytes on T. tabaci infesting onion were also examined. Seven fungal endophytes used in our study were able to colonize onion plants either by the seed or seedling inoculation methods. Seed inoculation resulted in 1.47 times higher mean percentage post-inoculation recovery of all the endophytes tested as compared to seedling inoculation. Fewer thrips were observed on plants inoculated with Clonostachys rosea ICIPE 707, Trichoderma asperellum M2RT4, Trichoderma atroviride ICIPE 710, Trichoderma harzianum 709, Hypocrea lixii F3ST1 and Fusarium sp. ICIPE 712 isolates as compared to those inoculated with Fusarium sp. ICIPE 717 and the control treatments. Onion plants colonized by C. rosea ICIPE 707, T. asperellum M2RT4, T. atroviride ICIPE 710 and H. lixii F3ST1 had significantly lower feeding punctures as compared to the other treatments. Among the isolates tested, the lowest numbers of eggs were laid by T. tabaci on H. lixii F3ST1 and C. rosea ICIPE 707 inoculated plants. These results extend the knowledge on colonization of onions by fungal endophytes and their effects on Thrips tabaci.

Quitosana na indução de resistência ao tombamento de plântulas de espécies olerícolas e no controle de fitopatógenos in vitro

Induction of resistance is defined as the activation of a state of resistance against diseases which is induced systemically in plants by the use of biotic or abiotic agents without any modification of the plant genome, occurring non-specific way, by activating genes coding for various plant defense responses. Chitosan is a polymer derived from the deacetylation of chitin, which is found in large quantities in crustacean shell, and studied with the potential to control plant pathogens, both by its direct fungistatic action, as the ability to induce protection of plants, indicating the presence of molecules of elicitoras characteristics. Three experiments with objective of evaluating the potential of chitosan in the seedling resistance induction were developed, beet (Beta vulgaris) seeds, cucumber (Cucumis sativus) seeds and tomato (Solanum lycopersicum) seeds, and the control of Fusarium sp., Rhizoctonia solani K¨uhn e Pythium sp. in vitro conditions. The experimental design was completely randomized, with four replications. Beet seeds, tomato and cucumber were submerged in chitosan solution for 20 minutes, in concentrations of 0.25, 0.5, 1 and 2% in the control and distilled water. Seeds were sown in trays containing Plantmax Florestalr substrate sterilized and inoculated with Fusarium sp., Rhizoctonia solani K¨unh and Pythium sp., respectively for the three cultures. The experiment was conducted for 14 days in growth chamber with controlled temperature (25 C 2 C), light (12 hour photoperiod) and humidity (70% 10%). The evaluations were seed emergency, seedling damping-off, seedling length, fresh weight and activity of the enzymes phenylalanine amˆonia-liase (PAL), chitinase and b-1,3-glucanase. It was also rated the mycelial growth of Fusarium sp., Pythium sp. and R. solani on P.D.A. (Potato-Dextrose and Agar) culture medium containing chitosan at the same concentrations evaluated in seeds. For beet growing, seed treatment with chitosan presented higher emergence and the length of the seedlings, and reduced the percentage of tipping. Treatment with chitosan activated the systemic acquired resistance with expression of chitinase and b-1,3-glucanase enzymes. For the tomato crop in chitosan concentration of 0.25% favored the emergency of seedlings, reduced the incidence of tipping and activated the PAL enzymes, chitinase and b-1,3-glucanase. In cucumber on the concentration of up 0.5% favored seedlings emergence and reduces the incidence of tipping. Chitosan activated the PAL enzymes and b-1,3-glucanase. Chitosan also presented fungistatic action on the initial growth of Pythium sp. and R. solani in vitro conditions, however, such action did not prevail until the end of the experiment. To Fusarium sp. the concentration of chitosan resulted in the reduction of mycelial growth in vitro.

Potencial do óleo de Aloysia citriodora Palau no controle de fitopatógenos e na indução de resistência ao tombamento de plântulas de feijão, pepino e beterraba

The crops are affected by pests and diseases that decrease productivity. Among them are the damping off of seedlings that can occur in pre and post-emergence. In bean crops, cucumber and beet these diseases occur, being caused by various pathogens, especialy fitopathogenic fungi. Several measures are used for the controle of such diseases, among them, is the chemical seed treatment fungicides. However, society has become increasingly concerned about the quality and food and environmental contamination, generation a growting search for sensitive products to humans and the environment. The use of essential oils to control plant pathogens is an example of alternative tested by science in the search for less aggressive technologies. This study aimed to evaluate the efficiency of the use of essential oil Aloysia citriodora, in control of pathogens causing damping off in beans, cucumber and beet. This thesis was divided in four chapters, the introductory first, and the other addressing the control of Pythium sp. in beans, Sclerotinia sclerotiorum on cucumber, and Fusarium sp. on beet. The methodology consisted of four experiments in each pathosystem, with all the work done at the Federal Technological University of Parana, Campus Dois Vizinhos. In the first experiment evaluated the fungistatic and fungicidal effect of the essential oil of A. citriodora on PDA in vitro in mycelial growth of pathogens studied. In the second experiment evaluated the in vitro effect of essential oil concentrations of A. citriodora in BD medium on microscope slides, on the germination of sporangia Pythium sp. and conidia Fusarium sp., and in Petri dishes with PDA medium, the sclerotia germination speed index of S. sclerotiorum. In the third experiment, we evaluated in germination test in paper roll (PR), the phytotoxic effect or not the use of essential oil concentrations of A. citriodora in dry bean seed, cucumber and beet. The variables used to assess this experiment were the germination percentage, mediun green mass per plant and average length of seedlings. In the fourth experiment we assessed the effect of treating bean seeds, cucumber and beet with essential oil contents of A. citriodora, seeds in their subsequent substrates contamined with pathogens studied, Pythium sp., S. sclerotiorum and Fusarium sp. In this experiment we used the following variables: percentage of emergence, percentage of post-emergence damping off, green average mass per plant, average length per plant and biochemical analyzes. The biochemistry of plant tissues evaluated were as follows: protein content, enzymatic activities of peroxidases, phenylalanine ammonia-liase (PAL), chitinases and β-1,3-glucanases. The in vitro results show that the essential oil has fungistatic and fungicidal effect on mycelial growth, on sporangia germination, conidia and sclerotia of the pathogens studied in this work, wich may be related to its major components, citral and limonene. The oil also exhibits low phytotoxicity to seeds of the species studied, only in beans decreases germination in most studied dosage (0,25%), cucumber also in the higher dosage (0,25%) reduce the length of seedlings, and beet there were no negative effects to the seedlings. In the test in substrate contaminated with the pathogens, the use of essential oil: increased germination and decreased post emergence damping off of beans seedlings; at a concentration of 0,0625% decreases post emergence damping off in cucumber. In biochemical analyzes found an increase in the enzymatic activity of peroxidases and β-1,3-glucanases on beans, and glucanases on cucumber, and increased enzyme activity of peroxidases on beet, showing action in resistance induction at damping off.

Indução de resistência e tratamento de sementes de soja com fosfitos de potássio

The use of inputs containing phosphites have been presenting results in many studies, taking on importance to the control of diseases in some cultures and demonstrating the resistance induction in seedlings, with ability to activate defense mechanisms, conferring protection to plants against microorganisms. The soybean crop is recognized for its importance in providing grains and derivatives for human consumption, animal, production of biofuels, pharmaceuticals, among others. Positive results obtained through studies based on resistance inducers in some cultures arouse the interest for further study. The objective of this study was to evaluate the effect of potassium phosphites on the resistance induction and treatment of soybean seeds. Therefore were conducted four laboratory studies at the Federal Technological University of Paraná, Campus of Dois Vizinhos. In the first study it was evaluated the quality attributes of the seeds and the resistance induction as seed treatment. Then it was verified that phosphites have action upon the seedlings metabolism in due to seed treatment, having the phosphite Reforce® contributed to seed quality attributes and phosphites FitofosK® and Fitofos K Plus® induced the resistance increasing the activity of β-1,3-glucanase. In the second study it was evaluated the the resistance induction in soybean cotyledons, in which the phosphites demonstrated induction potential of phytoalexin gliceolin. In the third study It was evaluated the soybean seed health treated with potassium phosphites.. it was observed that the phosphites reduced the incidence of many fungi on seeds, especially of storage fungi like Aspergillus sp. and Fusarium semitectum. In the fourth study it was evaluated the in vitro effect of potassium phosphites on pathogenic fungi of the culture. And it was found direct action of phosphites on the mycelial growth of Fusarium semitectum, Pythium sp. and Sclerotinia sclerotiorum. Based on these results, we concluded that potassium phosphites have potential in seeds treatment, as resistance inducer and on in vitro control of phytopathogens.

Isolation and recognition Saprolegnia species from fungal infected eggs of the rainbow trout in Mazandaran province farms

Fungal infection in the eggs of freshwater fish is well known as a problematic disease. That isolation and recognition Saprolegnia fungi from fungal infected eggs of the rainbow trout in Mazandaran province was the aim of this research. For this purpose fungal infected eggs were examined from six fish farm in the fall and winter 2005-2006. The eggs with fungi were inoculated on SDA, CMA, GPagar and hemp seed and sesame seed cultures in sterile tap water at room temperature (18-24°C). In this study recognized three genera and six species Saprolegniaceae members, based on morphological characteristics which contain: Saprolegnia, Achlya, Brevilegnia. Four species were identified in the genus Saprolegnia; S.mixta, S.parasitica, S.moniliphera, S.lapponica and one species was identified in the genus Achlya; A.oblongata. S.parasitica was isolated from almost all the farms. In addition, another nine genera and species were identified; Penicillium, Aspergillus, Paeciliomyces, Acremonium, Fusarium oxysporum, F.solani , Alternaria, Helminthosporium, Mucor.

Markers for seedling and adult plant crown rot resistance in four partially resistant bread wheat sources

QTL identified for seedling and adult plant crown rot resistance in four partially resistant hexaploid wheat sources. PCR-based markers identified for use in marker-assisted selection. Crown rot, caused by Fusarium pseudograminearum, is an important disease of wheat in many wheat-growing regions globally. Complete resistance to infection by F. pseudograminearum has not been observed in a wheat host, but germplasm with partial resistance to this pathogen has been identified. The partially resistant wheat hexaploid germplasm sources 2-49, Sunco, IRN497 and CPI133817 were investigated in both seedling and adult plant field trials to identify markers associated with the resistance which could be used in marker-assisted selection programs. Thirteen different quantitative trait loci (QTL) conditioning crown rot resistance were identified in the four different sources. Some QTL were only observed in seedling trials whereas others appeared to be adult plant specific. For example while the QTL on chromosomes 1AS, 1BS, and 4BS contributed by 2-49 and on 2BS contributed by Sunco were detected in both seedling and field trials, the QTL on 1DL present in 2-49 and the QTL on 3BL in IRN497 were only detected in seedling trials. Genetic correlations between field trials of the same population were strong, as were correlations between seedling trials of the same population. Low to moderate correlations were observed between seedling and field trials. Flanking markers, most of which are less than 10 cM apart, have now been identified for each of the regions associated with crown rot resistance.

A new species of gall midge (Diptera: Cecidomyiidae) feeding on ornamental Cordyline fruticosa(Asparagaceae) in Australia

Larvae of an undescribed gall midge were found feeding on leaves and stems within leaf sheaths and between leaf blades of potted plants of Cordyline fruticosa (Asparagaceae) in a production nursery in Queensland. The following varieties of the host plant were infested: Apple Blossom', Glauca', Kilauea', Negra', Pink Diamond, 'Purple Prince' and Willy's Gold'. The new species, Dasineura cordylineaeKolesik sp. nov., is described and its cytochrome oxidase unit I mitochondrial gene segment is sequenced. The new species is the first known gall midge feeding on a plant species of the genus Cordyline. Orange larvae induce oval shallow swellings on the leaf and stem tissue, which becomes necrotised during the later stage of larval feeding. Necrotic areas remain visible to the end of leaves' lives and decrease the market value of the plants. In the production nursery investigated, the lesions caused by the gall midge provided an entry for a fungal infection by Fusarium sp. inflicting further injury to plants. Larvae of the new species were preyed on by larvae of Gaurax sp. (Diptera: Chloropidae). This is the first worldwide record of Chloropidae preying on Cecidomyiidae.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains

In vitro experimental environments are used to study interactions between microorganisms, and predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to closely match the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation was studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25oC and 30oC). Competition experiments showed interaction between main effects of aflatoxin accumulation and environment at 25oC, but not so at 30oC. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-hour incubation in different experimental environments. Whereas, all fungi incubated within the soil environment survived; in the cotton-wool environment, none of the competitors of A. flavus survived at 30 oC. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post harvest.

What influences fungal communities in cotton soils

Soilborne diseases such as Fusarium wilt, Black root rot and Verticillium wilt have significant impact on cotton production. Fungi are an important component of soil biota with capacity to affect pathogen inoculum levels and their disease causing potential. Very little is known about the soil fungal community structure and management effects in Australian cotton soils. We analysed surface soils from ongoing field experiments monitoring cotton performance and disease incidence in three cotton growing regions, collected prior to 2013 planting, for the genetic diversity and abundance as influenced by soil type, environment and management practices and link it with disease incidence and suppression. Results from the 28S LSU rRNA sequencing based analysis indicated a total of 370 fungal genera in all the cotton soils and the top 25 genera in abundance accounted for the major portion of total fungal community. There were significant differences in the composition and genetic diversity of soil fungi between the different field sites from the three cotton growing regions. Results for diversity indices showed significantly greater diversity in the long-term crop rotation experiment at Narrabri (F6E) and experiments at Cowan and Goondiwindi compared to the Biofumigation and D1 field experiments at ACRI, Narrabri. Diversity was lowest in the soils under brassica crop rotation in Biofumigation experiment. Overall, the diversity and abundance of soil fungal community varied significantly in the three cotton growing regions indicating soil type and environmental effects. These results suggest that changes in soil fungal community may play a notable role in soilborne disease incidence in cotton.

Fumonisina B1 em arroz: validação de método e efeito de tratamento térmico nos níveis da micotoxina

O arroz (Oryza sativa, L.), como todos os cereais, pode ser contaminado por fungos responsáveis por danos tecnológicos, nutricionais e toxicológicos, dentre eles a produção de micotoxinas. Diversas toxinas fúngicas produzidas pelo gênero Fusarium tem sido relatadas em arroz, no entanto a fumonisina B1 (FB1) é pouco estudada neste grão. As principais características da FB1 é a alta solubilidade em solventes polares, estabilidade a altas temperaturas além de efeitos neurotóxicos e carcinogênicos. Assim o objetivo deste trabalho foi avaliar o efeito do tratamento térmico e hidrotérmico nos teores de fumonisina B1 e nas características químicas de arroz comercial. Na primeira etapa do trabalho foi adaptado um método para detecção e quantificação de FB1 em arroz cru e após cocção, por HPLC-FL. O método foi avaliado quanto aos indicativos de eficiência destacando-se o LOD (30 µg.kg-1) e a recuperação ( 90% para arroz cru e 86% pra arroz cozido). Na segunda etapa realizou-se o levantamento de ocorrência de FB1 em 05 diferentes amostras comerciais de arroz integral, branco e parboilizado da cidade de Rio Grande, RS, totalizando 9 amostras. Foi detectada a presença de FB1 em 7 das 9 amostras, sendo que os maiores índices foram encontrados em amostras de arroz parboilizado e integral apresentando níveis de contaminação entre 30 e 170 µg.kg-1. A terceira etapa do trabalho consistiu no estudo do efeito de tratamentos térmicos sobre os níveis de FB1 em amostras após aplicação de calor. Foram testados tratamento hidrotérmico com evaporação, tratamento hidrotérmico com autoclavagem e tratamento térmico seco. O maior nível de redução dos teores iniciais de FB1 foi 82,8% quando se empregou tratamento térmico seco a 125 °C/3 min. Ainda foram avaliados os efeitos do t ratamento hidrotérmico com evaporação de água na composição química e na digestibilidade protéica. Esta característica proporcionou aumento de até 100% na digestibilidade in vitro das proteínas e reduziu em média 73% do teor de contaminação com FB1.

Collecting banana diversity in eastern Indonesia

Major diseases, including Fusarium wilt tropical race 4, threaten banana production systems worldwide. New sources of genetic resistance are considered necessary in the fight against such diseases. The triangular region of Indonesia taking in Sulawesi, the Maluku Islands and Lesser Sunda Islands was prioritized by the Global Musa Genetic Resources Network, MusaNet for exploration and collecting. It is just east of the Wallace Line, which is recognized as a transition zone for flora in southeast Asia, and had been little explored. Bioversity International funded a team of scientists from Indonesia and Australia to make collecting missions in the triangle in October 2012 and February 2013. Suckers and seeds of 35 promising new accessions were collected. About 90% of these are either wild species or diploid cultivars of more direct use to breeding programs. These were morphologically characterized during the collecting missions and included a set of photographs recommended by Bioversitys Taxonomic Advisory Group. Cigar leaf samples were also collected and sent as fresh samples to the International Banana Genotyping Centre in the Czech Republic. Ploidy and DNA (SSR) genotyping determinations from these samples have been invaluable in quickly interpreting and better appreciating what has been discovered. The new accessions have been grown on at Solok field collection, West Sumatra and will be made available by Indonesia to the international community, including breeding programs, for evaluation and utilization. Information on wild Eumusa prompts a rethinking of the phytogeography of Musa acuminata. The variation within the Australimusa species M. lolodensis highlights the need for broader study of this Musa section. French Plantain-like edible AAs and prospects for the generation of African plantains in the region were identified. The mission indicated existence of local edible ABs in eastern Indonesia in association with balbisiana hybrids origins in the region. Further explorations in the region should add to Musa diversity knowledge.

Research into crown rot and wilt diseases of strawberries in Queensland

Plant losses due to fungal diseases in strawberry (Fragaria × ananassa Duch.) can potentially cause total loss of production. Three fungal pathogens, Fusarium oxysporum f. sp. fragariae, Colletotrichum gloeosporioides and Macrophomina phaseolina, cause similar crown rot and wilt symptoms in strawberries in Queensland. Since the withdrawal of methyl bromide in 2005, no effective chemical control of any of the three pathogens has been available. This study aims at identifying sources of plant genetic resistance that can be used in the breeding program to develop resistant cultivars for use as part of an integrated disease management plan in commercial strawberry production. Results from glasshouse pathogenicity and screening trials on the three pathogens showed that when breeding for resistance against a pathogen, resistance to other pathogens also needs to be considered, e.g., cultivar 'Festival' is resistant to F. oxysporum f. sp. fragariae, but is highly susceptible to C. gloeosporioides. The cultivars 'Earlisweet', 'Kabarla' and 'Phenomenal', two seedling clones and four DAFF breeding lines were resistant to C. gloeosporioides. Cultivar 'Suncoast Delight' showed the most promising level of resistance to M. phaseolina. These cultivars, breeding lines and seedling selections have been made available for incorporation into the crossing program to support the Queensland strawberry breeding program.

Analyses of the community compositions of root rot pathogenic fungi in the soybean rhizosphere soil

Soybean ( Glycine max [L.] Merr.) root rot is an important disease of soybean under continuous cropping, and root rot is widely distributed throughout the world. This disease is extremely harmful, and it is difficult to prevent and control. The study aimed to elucidate the composition of root rot pathogenic fungal communities in the continuous cropping of soybean. In this study, we employed PCRDGGE technology to analyze the communities of root rot pathogenic fungi in soybean rhizosphere soil subjected to continuous cropping during a season with a high incidence of root rot in Heilongjiang province, China, the main soybean producing area in China. The results of 13 DGGE bands were analyzed by phylogenetic revealed that the predominant root rot pathogenic fungi in rhizosphere soil in the test area were Pythium ultimum and Fusarium species. The results of cluster analysis showed that the duration of continuous cropping, the soybean variety and the plant growth stage all had significant effects on the diversity of root rot pathogenic fungi in rhizosphere soil.

Chemical profile and antifungal potential of essential oils from leaves and flowers of Salvia algeriensis (Desf.): A comparative study

Salvia is a plant genus widely used in folk medicine in the Mediterranean area since antiquity. A large number of Salvia essential oils have been reported against diverse microorganisms. In the current study, chemical composition of essential oils from leaves and flowers of Salvia algeriensis (Desf.) was determined using gas chromatography-electron impact mass spectrometry (GC-EIMS) as well as their antifungal activity against phytopathogenic fungi Alternaria solani and Fusarium oxysporum exploring disk method. The GC-EIMS analysis identified 59 compounds (84.8%) in the essential oil obtained from leaves of S. algeriensis. Its major constituents were benzaldehyde (9.7%), eugenol (8.7%) and phenylethyl alcohol (8.4%). In flowers oil, 34 compounds (92.8%) were detected. The main ones were viridiflorol (71.1%) and globulol (8.6%). The essential oil obtained from leaves exhibited the highest antifungal activity, where the effective dose inhibiting 50% of mycelial fungal (ED50) against A. solani was 0.90 μL mL-1 with minimum inhibitory concentration (MIC) equal to 2 μL mL-1, whereas the ED50 and MIC in F. oxysporum culture was 1.84 μL mL-1 and 3 μL mL-1 respectively. The mycelial inhibition by flowers oil varies from 1.77 μL mL-1 (ED50) with A. solani culture (MIC 6.5 μL mL-1) to the lowest effect recorded (ED50 3.00 μL mL-1 and MIC 9.33 μL mL-1) against F. oxysporum. To our best knowledge, this is the first report on S. algeriensis, their leaves oil can constitute an alternative biocontrol against phytopathogenic fungi commonly controlled by chemical fungicides.