Quantification of incubation, latent and infection periods of Phakopsora pachyrhizi in soybean, according to chronological time and degree-days

ABSTRACT In experiments conducted in a growth chamber, the chronological time and the accumulated degree-days were determined for the duration of incubation, latent and infectious periods of Phakopsora pachyrhizi cultivars BRSGO 7560 and BRS 246 RR. Detached soybean leaflets were placed in gerbox-type acrylic boxes and inoculated with 20 x 103 uredospores/mL. The study was conducted at 12-h photoperiod and temperatures of 10ºC, 15ºC, 22ºC, 25ºC and 30°C for 30 days. Lesions and uredia/cm2were evaluated and the number of uredia per lesion was quantified after the beginning of sporulation. The sporulation potential was also quantified for cultivars BRSGO 7560 and BRS 246 RR. The steps of the infection process can be quantified based on both the chronological time and the accumulated heat. The cultivar BRSGO 7560 produced 4,012.8 spores/cm2 and BRS 246 RR, 7,348.4 uredospores/cm2. The largest number of uredia was produced at 25ºC in both cultivars; however, BRS 246 RR presented 372.7 uredia/cm2 and BRSGO 7560, 231.6 uredia/cm2. At 10ºC and 30°C, leaf infection did not occur in both cultivars.

PLANT GROWTH-PROMOTING MICROBIAL INOCULANT FOR Schizolobium parahyba pv. parahyba

ABSTRACTSchizolobium parahyba pv. amazonicum (Huber ex Ducke) Barneby (paricá) occurs naturally in the Amazon and is significant commercial importance due to its rapid growth and excellent performance on cropping systems. The aim of this paper was to evaluate a microbial inoculants such as arbuscular mycorrhiza fungi (AMF) and Rhizobium sp. that promote plant growth. The inocula was 10 g of root colonized and spores of Glomus clarum and/or 1 mL of cell suspension (107 CFU/mL) of Rhizobium sp. and/or 100 g of chemical fertilizer NPK 20-05-20 per planting hole. The experimental design was complete randomized blocks with five replications and eight treatments (n = 800). Plant height, stem diameter and plant survival were measured. The results were tested for normality and homogeneity of variances and analyzed by ANOVA and Tukey test (p < 0.05). Rhizobium sp and AM fungi showed no effect on plant growth. Environmental factors probably influenced the effectiveness of symbiosis of both microorganisms and plant growth. The chemical fertilizer increased S. parahyba growth. During the first 120 days plants suffered with drought and frost, and at 180 days plants inoculated with microorganism plus chemical fertilizer showed higher survival when compared with control. The results showed that the microbial inoculants used showed an important role on plant survival after high stress conditions, but not in plant growth. Also was concluded that the planting time should be between November to December to avoid the presence of young plants during winter time that is dry and cold.

EVALUATION OF SUBSTRATES AND AMF SPORULATION IN THE PRODUCTION OF SEEDLINGS OF NATIVE FOREST SPECIES

ABSTRACT The objective of this study was to evaluate organic substrates in the production of canafistula (Peltophorum dubium) (Spreng.) Taub, cutieira (Joannesiaprinceps Vell.), jatoba (Hymenaea courbaril L.) and rubber tree (Hevea brasiliensis M. Arg.) seedlings, native trees with potential use in forest restoration programs. The design was completely randomized with 10 substrate formulations with 4 repetitions of 3 plants for the four species. The evaluated substrates consisted of soil, bovine manure (BM), poultry manure (PM), chemical fertilizer (CF) and sand, in different proportions. The experiment was concluded at the end of 180 days for canafistula, cutieira and rubber and 210 days for jatoba. At the end of these periods, the root (RDM), shoot (SDM) and total (TDM) the dry matters of the seedlings were determined. Quantification of AMF spores and normalization between samples through SPORES/RDM correction were also performed. The Scott-Knott test at 5% probability was applied. Regarding biomass production, only canafistula had significant difference among the tested substrates. In relation to sporulation, the highest values were observed in cutieira and rubber tree in substrate containing PM. The substrates composed of 40 or 50% soil + 20% sand + 30% or 40 PM for canafistula; 50% soil + 20% sand + 30% PM for cutieira; and for jatoba and rubber tree 60% soil + 20% sand + 20% PM, enabled the best results in terms of biomass production in seedlings and AMF sporulation.

Crystal-associated cholangiopathy in sheep grazing Brachiaria decumbens containing the saponin protodioscin

An outbreak of hepatogenous photosensitization is reported in a flock of 28 sheep grazing Brachiaria decumbens in Mato Grosso do Sul State, Central-Western Brazil. Seven lambs and an adult sheep were affected and 6 of them died. Two surviving affected lambs and one lamb without clinical signs had increased serum values of gamma glutamyltransferase, bilirubin, and cholesterol. In two adult unaffected sheep those parameters were within normal values. An adult sheep submitted to necropsy presented moderate body condition, unilateral corneal opacity, drying of the muzzle, moderate jaundice, increased lobular pattern of the liver, and a distended gallbladder. Histological lesions were epithelial degeneration, necrosis, and hyperplasia of small bile ducts. Mild amounts of foamy macrophages were observed, mainly in the centroacinar zone. Diffuse swelling and vacuolation were observed in hepatocytes. Crystal negative images were found within bile ducts, foamy macrophages, and the lumen of some renal tubules. The heart showed multifocal areas of degeneration and necrosis of the muscle fibers. Pasture samples (Brachiaria decumbens) contained 2.36% of protodioscin. No Pithomyces chartarum spores were found in the pasture. Samples from a similar neighboring B. decumbens pasture grazed by cattle without photosensitization contained 1.63% of protodioscin isomers. Outbreaks of photosensitization caused by Brachiaria spp. are common in cattle in the Brazilian Cerrado (savanna) with about 51 million hectares of Brachiaria spp pastures. Sheep farming has been recently developed in this region, and the number of sheep is increasing significantly. Because sheep are more susceptible than cattle to lithogenic saponins, poisoning by Brachiaria should be an important limiting factor for the sheep industry.

Myxosporidiosis in intensively-reared Piaractus mesopotamicus: Histopathological diagnosis by means of Ziehl-Neelsen staining

Samples of different organs from intensively-reared Piaractus mesopotamicus were collected and processed using routine histological techniques in order to produce thin sections for staining with hematoxylin-eosin and with the Ziehl-Neelsen method. Through examination under an optical microscope, myxosporidians of the genera Henneguya sp. and Myxobolus sp. were identified, respectivelyin the gills and kidneys of P. mesopotamicus. Plasmodia with immature spores of Henneguya sp. were located along the secondary lamellae, with total length of 30.45±4.84µm and width of 3.52±0.33µm. Spores of Myxobolus sp. were located in the kidneys, with total length of 8.94±0.82µm and width of 5.59±0.39µm. Histopathological analysis of the gills showed plasmodia containing spores of Henneguya sp., at intralamellar and intravascular localities, at different stages of development. Spores of Myxobolus sp. were identified in the kidneys, in the peritubular region and in the interstices and glomerulus, surrounded by melanomacrophages. Focal hemorrhage was recorded in a few cases. Ziehl-Neelsen staining allowed to identify particular features of the spores and facilitated biometry and enabled classification in comparison with hematoxylin-eosin, thus demonstrating its usefulness for histopathological diagnosis of the parasitosis.

Effects of using different host plants on the detected biodiversity of arbuscular mycorrhizal fungi from an agroecosystem

The influence of peanut (Arachis hypogaea L.), sorghum (Sorghum bicolor (L.) Moench) and maize (Zea mays L.) on the development and diversity of arbuscular mycorrhizal fungi (AMF) from an agrosystem was investigated. Soil collected from an agricultural field where maize had been grown was inserted into sowing holes, under the seeds of peanut, sorghum and maize those were subsequently grown in sterilised quartz sand separately in plastic boxes for five months. After this period, collections of roots and rhizospheric soil were made to evaluate the percentages of root colonization (RC), number of spores (NS) and species of AMF. Peanut showed the highest average values for RC and NS: 24.5% and 547.8/100 g of soil, respectively. Maize had an average RC of 19.7% and 415.2 spores/100g soil. Sorghum showed the lowest values: 15.9% for average RC and 349.8 spores/100 g soil. A total of fourteen species of AMF were identified. Seven species were identified from peanut rhizospheres, Entrophospora colombiana being the most abundant and frequent. In sorghum rhizospheres, twelve species were found, Glomus geosporum was the dominant taxon in terms of number of spores and frequency. Ten species were detected in maize with Acaulospora longula being the most abundant and the most frequent. It was observed that peanut was the best plant for promoting the sporulation of AMF, while sorghum favoured the establishment of most AMF species, followed by maize.

Biflavonoids inhibit the production of aflatoxin by Aspergillus flavus

The biflavonoids 6,6"-bigenkwanin, amenthoflavone, 7,7"-dimethoxyagastisflavone and tetradimethoxybigenkwanin isolated from Ouratea species were tested for inhibitory activity on Aspergillus flavus cultures. Suspensions of Aspergillus flavus spores were inoculated into 50 ml of YES medium at different biflavonoid concentrations: 5 and 10 µg/ml for 6,6"-bigenkwanin, amenthoflavone and 7,7"-dimethoxyagastisflavone, and 5, 10, 15 and 20 µg/ml for tetradimethoxybigenkwanin. The four biflavonoids showed inhibitory activity on aflatoxin B1 and B2 production (P<0.001), but did not inhibit fungal growth at the concentration tested (P>0.05). These results show that biflavonoids can be used for the development of agents to control aflatoxin production.

Survey on ochratoxin A in Brazilian green coffee destined for exports

The presence of ochratoxin A (OTA) in foods has led some countries to establish regulatory limits. Although coffee is not a major source of OTA in human consumption, the European Community (EC) may establish limits in the near future, with possible economic impact on producing countries. This study measured the OTA content with HPLC in 37 samples of Brazilian green coffee exclusive destined to the export market and also verified a possible relation between coffee defects and OTA content. The results showed an OTA concentration ranging from < 0.16ng/g (detection limit) to 6.24ng/g (average of 3.20ng/g) for 37 samples. Of the five samples observed for defects, toxin content of sound beans ranged from 0.22 to 0.80ng/g (average 0.46ng/g) and of defective beans from 0.42 to 17.46 (average 4.52ng/g). Morphological differences among sound and defective beans showed no susceptibility for mould invasion under optical microscopy observation. One black bean depicted the presence of mould and spores on observation under Scanning Electron Microscope (SEM). According to this investigation, Brazilian green coffee for export complies with most limits in place.

Biotransformation of citronellol in rose-oxide using cassava wastewater as a medium

The use of liquid cassava waste (manipueira) as the medium for the biotransformation of citronellol using a Penicillium sp strain was studied. The strain was able to grow in the waste and production of cellular mass reaching 25 g/L over three days of contact of the spores with the medium. Submerged cultures of Penicillium sp grown in manipueira were able to convert the substrate into cis- and trans-rose oxides when the cells were transferred into a mineral medium for the biotransformation experiments. The production of rose oxide increased by more than 2.4 times using this 2 media process as compared to processes using only a manipueira medium (cassava medium). Auto-oxidation products were not detected in the control experiments.

Introducing the concept of critical Fo in batch heat processing

The determination of the sterilization value for low acid foods in retorts includes a critical evaluation of the factory's facilities and utilities, validation of the heat processing equipment (by heat distribution assays), and finally heat penetration assays with the product. The intensity of the heat process applied to the food can be expressed by the Fo value (sterilization value, in minutes, at a reference temperature of 121.1 °C, and a thermal index, z, of 10 °C, for Clostridium botulinum spores). For safety reasons, the lowest value for Fo is frequently adopted, being obtained in heat penetration assays as indicative of the minimum process intensity applied. This lowest Fo value should always be higher than the minimum Fo recommended for the food in question. However, the use of the Fo value for the coldest can fail to statistically explain all the practical occurrences in food heat treatment processes. Thus, as a result of intense experimental work, we aimed to develop a new focus to determine the lowest Fo value, which we renamed the critical Fo. The critical Fo is based on a statistical model for the interpretation of the results of heat penetration assays in packages, and it depends not only on the Fo values found at the coldest point of the package and the coldest point of the equipment, but also on the size of the batch of packages processed in the retort, the total processing time in the retort, and the time between CIPs of the retort. In the present study, we tried to explore the results of physical measurements used in the validation of food heat processes. Three examples of calculations were prepared to illustrate the methodology developed and to introduce the concept of critical Fo for the processing of canned food.

Origanum vulgare L. essential oil as inhibitor of potentially toxigenic Aspergilli

Origanum vulgare L. essential oil has been known as an interesting source of antimicrobial compounds to be applied in food conservation. In this study, the effect of O. vulgare essential on the growth of A. flavus, A. parasiticus, A. fumigatus, A. terreus and A. ochraceus was assessed. The essential oil had a significant inhibitory effect on all assayed fungi. MIC was 0.6 µL.mL-1 for all fungi, while MFC was in the range of 1.25-2.5 µL.mL-1. The radial mycelial growth of A. flavus and A. parasiticus was strongly inhibited over 14 days at 0.6, 1.25 and 2.5 µL.mL-1 of oil in solid medium. The mycelial mass of all fungi was inhibited over 90% at 0.6 and 0.3 µL.mL-1 in liquid medium, while it was 100% at 1.25 µL.mL-1. The oil in a range of concentrations (0.6 to 2.5 µL.mL-1) was effective in inhibiting the viability and spores germination in a short time of exposure. The main morphological changes caused by the essential oil in A. parasiticus observed under light microscopy were absence of conidiation, leakage of cytoplasm, loss of pigmentation, and disrupted cell structure. These results demonstrated that O. vulgare essential oil produced a significant fungitoxic effect supporting its possible rational use as anti-mould compound in food conservation.

Adhesion of uredospores of Phakopsora pachyrhizi on soybean seeds and their viability during storage

To study adhesion and viability of uredospores of the fungus Phakopsora pachyrhizi on soybean seeds during storage, suspension tests of those uredospores were carried out by washing seeds at each 30 days interval. Furthermore, germination and inoculation tests of uredospores on soybean plants were performed with uredospores collected from seeds of two soybean production areas, located in the municipalities "Chapada dos Guimarães" and "Tangará da Serra", State of Mato Grosso, Brazil. High levels of uredospores infestation were detected before storage [249.31 and 85.18 uredospores/100 seeds (U/100)] on seeds collected in both localities, respectively. After 30 days storage, these figures were 46.12 and 122.5 U/100; at 60 days were 14.62 and 26.62 U/100; and at 90 days were only 2.87 and 3,68 U/100, respectively; dropping to zero after 120 days storage. The percentage of germinated uredospores decreased with increasing storage periods and at 120 days germination percentage was nil. When uredospores were inoculated on soybean plants, rust symptoms were only observed for uredospores collected from freshly harvested seeds. Uredospores associated to soybean seed germinate until 90 days after storage, but are not viable after this time span. Infection of plants only occurs with inoculation of uredospores obtained from freshly harvested seeds.