Characterization of nontypable haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

In vitro activity of essential oils of Lippia sidoides and Lippia gracilis and their major chemical components against Thielaviopsis paradoxa, causal agent of stem bleeding in coconut palms

Essential oils of Lippia sidoides, Lippia gracilis and their main chemical components were investigated for in vitro control of Thielaviopsis paradoxa. Mycelial growth and a number of pathogen conidia were inhibited by the essential oil of L. sidoides at all concentrations tested (0.2; 0.5; 1.0; 3.0 µL mL-1). L. sidoides oil contained 42.33% thymol and 4.56% carvacrol, while L. gracilis oil contained 10% thymol and 41.7% carvacrol. Mycelial growth and conidial production of T. paradoxa were completely inhibited by thymol at a 0.3 µL m-1 concentration. The results suggest that thymol could potentially be used for controlling coconut stem bleeding.

Phytotoxic effects of metabolites from Alternaria euphorbiicola against its host plant Euphorbia heterophylla

A bioassay-guided fractionation of culture filtrates of the fungus Alternaria euphorbiicola, a pathogen of the weed Euphorbia heterophylla, led to the isolation of anhydromevalonolactone (1), tyrosol (2), (R)-( - )-mevalonolactone (3), and cycloglycylproline (4). When tested on the punctured leaves of the host plant, these compounds produced bleached lesions with dark brown margins at concentrations as low as 80 µM. When tested on the leaves of other relevant weeds, only cycloglycylproline showed selective activity against E. heterophylla. This is the first report on the isolation of phytotoxins from A. euphorbiicola and on the phytotoxicity of anhydromevalonolactone, (R)-( - )-mevalonolactone, and cycloglycylproline.

Reaction of sorghum genotypes to the anthracnose fungus Colletotrichum graminicola

The reactions of 22 sorghum (Sorghum bicolor) genotypes to six previously identified races of the sorghum anthracnose fungus Colletotrichum graminicola, were evaluated under greenhouse and field conditions. Races were inoculated in separate tests in the greenhouse. In the field, spreader rows of a susceptible genotype were artificially inoculated with a mixture of the six races of the pathogen. In the greenhouse tests, nine genotypes showed resistance to all six races. In the field high levels of dilatory resistance was observed in the sorghum genotypes CMSXS169 and CMSXS373.

Differences in competitive ability among races of Colletotrichum graminicola in mixtures

Mixtures of races of Colletotrichum graminicola, causing sorghum (Sorghum bicolor) anthracnose and differing in their virulence range, were inoculated for five and six generations on the susceptible sorghum cultivar BR009 (Tx623), in two experiments in a greenhouse. In each generation a sample of 50 single spore isolates was obtained and inoculated on a standard differential set to determine the proportion of each race in the mixture. Isolates of the race 30A, with the narrowest virulence range, predominated over isolates of the more complex races 31B, 31C and 31E indicating the existence of differences in the survival ability among races of this pathogen.

Concepts in plant disease resistance

Resistance to nearly all pathogens occurs abundantly in our crops. Much of the resistance exploited by breeders is of the major gene type. Polygenic resistance, although used much less, is even more abundantly available. Many types of resistance are highly elusive, the pathogen apparently adapting very easily them. Other types of resistance, the so-called durable resistance, remain effective much longer. The elusive resistance is invariably of the monogenic type and usually of the hypersensitive type directed against specialised pathogens. Race-specificity is not the cause of elusive resistance but the consequence of it. Understanding acquired resistance may open interesting approaches to control pathogens. This is even truer for molecular techniques, which already represent an enourmously wide range of possibilities. Resistance obtained through transformation is often of the quantitative type and may be durable in most cases.

Epidemiology of apple leaf spot

Apple leaf spot (ALS) caused by Colletotrichum spp. is a major disease of apple (Malus domestica) in Southern Brazil. The epidemiology of this disease was studied in experiments carried out in the counties of Passo Fundo and Vacaria, State of Rio Grande do Sul, from February 1998 to October 2000. The disease was found in all the six apple orchards sampled in the growing seasons of 1997/98 and 1998/99. The fungus isolates associated with ALS fit the characteristics of C. gloeosporioides (75%), C. acutatum (8%), and Colletotrichum sp. (17%). The pathogen overwintered in dormant buds and twigs but not in dropped leaves or fruit mummies. Two sprays of copper oxychloride (at 0.3%) reduced the fungus initial inoculum by 65-84.6% in buds and 85.6-93.7% in twigs, but had no effect on the early season progress of the disease. Disease severity increased proportionally to elevation of temperature from 14 to 26-28 °C. At 34 °C, however, infection was completely inhibited. The duration of leaf wetness required for infection ranged from two hours at 30 °C to 32 h at 16 °C. The relationship of temperature (T) and leaf wetness (W) to disease severity (Y) was represented by the model equation Y = 0.00145[((T-13)1.78)((34.01-T )1.09)] * 25/[1+14 exp(-0.137W)], R² = 0.73 and P < 0.0001. Currently, this information is being used to manage the disease and to validate a forecast system for ALS.

Variability in isolates of Puccinia polysora in Brazil

The main objective of this work was to evaluate the variability of the southern rust pathogen Puccinia polysora in Brazil, based on its virulence on a set of maize (Zea mays) cultivars. Sixty single pustule isolates, from different areas of occurrence of southern rust, were evaluated for their virulence to 50 maize experimental hybrids. Six cultivars showed a clear distinction between susceptible and resistant reaction, and were used to characterize the variability of the pathogen. Seventeen virulence patterns were identified among the 60 isolates tested. The most frequent virulence patterns identified, were observed in all locations of sampling, which suggests the absence of geographical differentiation among prevalent populations of P. polysora in Brazil.

Phytophthora palmivora causando podridão de frutos de mamoeiro no Pará

Phytophthora palmivora was isolated from papaya (Carica papaya) fruit rots, from commercial growth in Capanema County, Pará. The fungus was pathogenic to healthy inoculated fruits and reisolated, fullfiling the Koch postulates. This is the first report of this pathogen on papaya in Pará, Brazil.

Pathotype diversity of Pyricularia grisea from improved upland rice cultivars in experimental plots

A study was undertaken to examine the pathogenic diversity of Pyricularia grisea isolates retrieved from 14 upland rice (Oryza sativa) cultivars in experimental plots during a period of five years. Inoculations were performed on 32 genotypes with 85 monoconidial isolates under controlled greenhouse conditions. Based on the reaction pattern of eight international differentials, eleven pathotypes of P. grisea were identified. The predominant international races or pathotypes were IB-9 (56.4%), IB-1 (16.4%) and IB-41 (11.8%). A set of eight commercial upland rice cultivars ('Carajás', 'Confiança', 'Maravilha', 'Primavera', 'Progresso', 'Caiapó', 'IAC-47', 'IAC-201') was utilized as additional differentials for describing the virulence pattern of P. grisea. Twenty-six Brazilian pathotypes were identified on the basis of disease reaction on these differentials, in contrast to the 11 international pathotypes. The most predominant Brazilian pathotypes, BB-21 and BB-41 were represented by 28.2% and 17.6% of the isolates tested, respectively. Isolates virulent and avirulent to cultivar 'Primavera' were encountered within the pathotype IB-1. Utilizing Brazilian cultivars as differentials, the 14 isolates of the pathotype IB-1could be further classified into eight local pathotypes, BB-41, BB-13, BB-21, BB-9, BB-29, BB-61, BD-9 and BG-1. Virulence to improved rice cultivars 'Canastra', 'Confiança', 'Carisma', 'Maravilha', 'Primavera' and 'Bonança' was frequent in pathogen population. Some of the Brazilian pathotypes that showed differential reaction on commercial rice cultivars could be utilized for incorporating resistance genes in susceptible cultivars improved for grain quality, by conventional breeding methods.

Podridão basal pós-colheita do coco verde no estado do Ceará

A new post-harvest disease of coconut (Cocos nucifera) was recently detected in fruits exported to European countries. The main symptoms are the blacking and cracking of basal parts of the fruits. Water oozing may occurs as the infection progresses. The fungus Lasiodiplodia theobromae has been frequently isolated from the lesions. Inoculation tests proved that this pathogen is the causal agent of the coconut fruit basal rot. This is the first occurrence of this post-harvest disease in Brazil.

Genotypic diversity among brazilian isolates of Macrophomina phaseolina revealed by RAPD

Macrophomina phaseolina has been considered one of the most prevalent soybean (Glycine max) pathogens in Brazil. No genetic resistance has been determined in soybean and very little is known about the genetic diversity of this pathogen in tropical and sub-tropical regions. Fifty-five isolates from soybean roots were collected in different regions and analyzed through RAPD for genetic diversity. The UPGMA cluster analysis for 74 loci scored permitted identification of three divergent groups with an average similarity of 99%, 92% and 88%, respectively. The three groups corresponded to 5.45%, 59.95% and 34.6%, respectively of all isolates used. A single plant had three different haplotypes, while 10.9% of the analyzed plants had two different haplotypes. In another study the genetic similarity was evaluated among isolates from different hosts [soybean, sorghum (Sorghum bicolor), sunflower (Helianthus annuus), cowpea (Vigna unguiculata), corn (Zea mays) and wheat (Triticum aestivum)] as well as two soil samples from native areas. Results showed that more divergent isolates originated from areas with a single crop. Isolates from areas with crop rotation were less divergent, showing high similarity values and consequently formed the largest group. Amplification of the ITS region using primers ITS1 and ITS4 produced only one DNA fragment of 620 bp. None of the isolates were differentiated through PCR-RFLP. Our results demonstrated genetic variability among Brazilian isolates of M. phaseolina and showed that one single root can harbor more than one haplotype. Moreover, cultivation with crop rotation tends to induce less specialization of the pathogen isolates. Knowledge of this variation may be useful in screening soybean genotypes for resistance to charcoal rot.

Cytogenetics of Colletotrichum lindemuthianum (Glomerella cingulata f. sp. phaseoli)

Cytogenetic and morphological studies were conducted with Colletotrichum lindemuthianum (Glomerella cingulata f. sp. phaseoli), the pathogen responsible for anthracnose of common bean (Phaseolus vulgaris). In this species, there is some evidence of genomic variation but it is unknown whether the process occurs in a manner similar to other fungal genetic models. Six isolates from bean plants were used and sexual reproduction was observed in vitro. Meiosis and ascospore formation were investigated by cytogenetical approaches and light microscopy. To study the nucleus and chromosome numbers, a mixture of carmine and orcein propionic dyes was used. Nucleus divisions as well as ascospore maturation were asynchronous. Meiosis was observed in three isolates. In the asexual form, chromosomal polymorphism in conidia was also observed microscopically and the mitosis process was described.

Production of caffeine alkaloid by Claviceps sorghi

The ergot disease of sorghum (Sorghum bicolor), caused by the fungus Claviceps sorghi, restricted to the Indian sub-continent, is a disease in which the pathogen infects the florets, colonizing the unfertilized ovaries. Losses are higher in hybrid seed production fields due to a higher susceptibility of male sterile lines. The sclerotia of C. sorghi have never been found to contain alkaloids with a tetracyclic ergoline ring system, which is normal in most ergot pathogens. In this work, we show that sclerotia of C. sorghi contain caffeine alkaloid and the ability to produce it in vitro.

Effect of organic amendments on sclerotial germination, mycelial growth, and Sclerotium rolfsii-induced diseases

The addition of organic residues to soil is an option to control some soil-borne diseases. Benzaldehyde and powders of kudzu (Pueraria lobata), velvetbean (Mucuna deeringiana), and pine-bark (Pinus elliottii and P. taeda) added to soil could reduce certain soil-borne diseases. This study evaluated the effects of benzaldehyde and the dried powders of kudzu, velvetbean, and pine-bark as soil amendments on germination and formation of sclerotia, on mycelial growth of Sclerotium rolfsii, on plant survival, and disease incidence. The data showed that high amounts of benzaldehyde (0.4 ml kg-1 of soil) and velvetbean (100 g kg-1) inhibited S. rolfsii mycelial growth and sclerotium germination. However, low amounts of benzaldehyde (0.1 ml kg-1), kudzu (25 g kg-1), and pine-bark (25 g kg-1) stimulated mycelial growth and sclerotium germination. Kudzu (25-100 g kg-1) and velvetbean (25-100 g kg-1) inhibited the formation of sclerotia. Nevertheless, benzaldehyde at 0.2 and 0.4 ml kg-1 stimulated the formation of sclerotia. Kudzu (50 and 100 g kg-1) and pine-bark (50 g kg-1) favored the colonization of sclerotia by Trichoderma sp. The numbers of soybean (Glycine max) plants were higher and diseased plants were lower than the non-amend soil in the following treatments: kudzu (50 and 100 g kg-1), velvetbean (50 and 100 g kg-1), and pine-bark (50 g kg-1). Disease severity on tomato (Lycopersicon esculentum) plants was low in soil treated with kudzu or velvetbean (30 and 35 g kg-1) and pine-bark (35 g kg-1). Dried powders of kudzu, velvetbean, or pine-bark added to soil can reduce disease by reducing pathogen inoculum.