Development of molecular monitoring methods and assessment of the environmental fate of the biological control agent of fire blight Pseudomonas fluorescens EPS62e

Pseudomonas fluorescens EPS62e es va seleccionar com a agent de biocontrol del foc bacterià per la seva eficàcia en el control de Erwinia amylovora. En aquest treball es van desenvolupar mètodes de traçabilitat que van permetre la seva detecció específica i quantificació. Mitjançant les tècniques RAPD i U-PCR es van obtenir fragments d'amplificació diferencial per EPS62e que es van seqüenciar i caracteritzar com marcadors SCAR per dissenyar una PCR en temps real. La PCR a temps real es va utilitzar simultàniament amb mètodes microbiològics per estudiar l'adaptabilitat epifítica de EPS62e en pomera i perera. L'ús combinat de mètodes microbiològics i moleculars va permetre la identificació de tres estats fisiològics de EPS62e: la colonització activa, l'entrada en un estat de viable però no cultivable, i la mort cel·lular. Aquest treball mostra que EPS62e està ben adaptada a la colonització de flors a camp, encoratjant la seva utilització dins d'una estratègia de control biològic contra el foc bacterià.

Development of molecular-based techniques for the detection, identification and quantification of food-borne pathogens

La presencia de microorganismos patógenos en alimentos es uno de los problemas esenciales en salud pública, y las enfermedades producidas por los mismos es una de las causas más importantes de enfermedad. Por tanto, la aplicación de controles microbiológicos dentro de los programas de aseguramiento de la calidad es una premisa para minimizar el riesgo de infección de los consumidores. Los métodos microbiológicos clásicos requieren, en general, el uso de pre-enriquecimientos no-selectivos, enriquecimientos selectivos, aislamiento en medios selectivos y la confirmación posterior usando pruebas basadas en la morfología, bioquímica y serología propias de cada uno de los microorganismos objeto de estudio. Por lo tanto, estos métodos son laboriosos, requieren un largo proceso para obtener resultados definitivos y, además, no siempre pueden realizarse. Para solucionar estos inconvenientes se han desarrollado diversas metodologías alternativas para la detección identificación y cuantificación de microorganismos patógenos de origen alimentario, entre las que destacan los métodos inmunológicos y moleculares. En esta última categoría, la técnica basada en la reacción en cadena de la polimerasa (PCR) se ha convertido en la técnica diagnóstica más popular en microbiología, y recientemente, la introducción de una mejora de ésta, la PCR a tiempo real, ha producido una segunda revolución en la metodología diagnóstica molecular, como pude observarse por el número creciente de publicaciones científicas y la aparición continua de nuevos kits comerciales. La PCR a tiempo real es una técnica altamente sensible -detección de hasta una molécula- que permite la cuantificación exacta de secuencias de ADN específicas de microorganismos patógenos de origen alimentario. Además, otras ventajas que favorecen su implantación potencial en laboratorios de análisis de alimentos son su rapidez, sencillez y el formato en tubo cerrado que puede evitar contaminaciones post-PCR y favorece la automatización y un alto rendimiento. En este trabajo se han desarrollado técnicas moleculares (PCR y NASBA) sensibles y fiables para la detección, identificación y cuantificación de bacterias patogénicas de origen alimentario (Listeria spp., Mycobacterium avium subsp. paratuberculosis y Salmonella spp.). En concreto, se han diseñado y optimizado métodos basados en la técnica de PCR a tiempo real para cada uno de estos agentes: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, y también se ha optimizado y evaluado en diferentes centros un método previamente desarrollado para Salmonella spp. Además, se ha diseñado y optimizado un método basado en la técnica NASBA para la detección específica de M. avium subsp. paratuberculosis. También se evaluó la aplicación potencial de la técnica NASBA para la detección específica de formas viables de este microorganismo. Todos los métodos presentaron una especificidad del 100 % con una sensibilidad adecuada para su aplicación potencial a muestras reales de alimentos. Además, se han desarrollado y evaluado procedimientos de preparación de las muestras en productos cárnicos, productos pesqueros, leche y agua. De esta manera se han desarrollado métodos basados en la PCR a tiempo real totalmente específicos y altamente sensibles para la determinación cuantitativa de L. monocytogenes en productos cárnicos y en salmón y productos derivados como el salmón ahumado y de M. avium subsp. paratuberculosis en muestras de agua y leche. Además este último método ha sido también aplicado para evaluar la presencia de este microorganismo en el intestino de pacientes con la enfermedad de Crohn's, a partir de biopsias obtenidas de colonoscopia de voluntarios afectados. En conclusión, este estudio presenta ensayos moleculares selectivos y sensibles para la detección de patógenos en alimentos (Listeria spp., Mycobacterium avium subsp. paratuberculosis) y para una rápida e inambigua identificación de Salmonella spp. La exactitud relativa de los ensayos ha sido excelente, si se comparan con los métodos microbiológicos de referencia y pueden serusados para la cuantificación de tanto ADN genómico como de suspensiones celulares. Por otro lado, la combinación con tratamientos de preamplificación ha resultado ser de gran eficiencia para el análisis de las bacterias objeto de estudio. Por tanto, pueden constituir una estrategia útil para la detección rápida y sensible de patógenos en alimentos y deberían ser una herramienta adicional al rango de herramientas diagnósticas disponibles para el estudio de patógenos de origen alimentario.

Iron Uptake and Homeostasis in Microorganisms

Preface. Iron is considered to be a minor element employed, in a variety of forms, by nearly all living organisms. In some cases, it is utilised in large quantities, for instance for the formation of magnetosomes within magnetotactic bacteria or during use of iron as a respiratory donor or acceptor by iron oxidising or reducing bacteria. However, in most cases the role of iron is restricted to its use as a cofactor or prosthetic group assisting the biological activity of many different types of protein. The key metabolic processes that are dependent on iron as a cofactor are numerous; they include respiration, light harvesting, nitrogen fixation, the Krebs cycle, redox stress resistance, amino acid synthesis and oxygen transport. Indeed, it is clear that Life in its current form would be impossible in the absence of iron. One of the main reasons for the reliance of Life upon this metal is the ability of iron to exist in multiple redox states, in particular the relatively stable ferrous (Fe2+) and ferric (Fe3+) forms. The availability of these stable oxidation states allows iron to engage in redox reactions over a wide range of midpoint potentials, depending on the coordination environment, making it an extremely adaptable mediator of electron exchange processes. Iron is also one of the most common elements within the Earth’s crust (5% abundance) and thus is considered to have been readily available when Life evolved on our early, anaerobic planet. However, as oxygen accumulated (the ‘Great oxidation event’) within the atmosphere some 2.4 billion years ago, and as the oceans became less acidic, the iron within primordial oceans was converted from its soluble reduced form to its weakly-soluble oxidised ferric form, which precipitated (~1.8 billion years ago) to form the ‘banded iron formations’ (BIFs) observed today in Precambrian sedimentary rocks around the world. These BIFs provide a geological record marking a transition point away from the ancient anaerobic world towards modern aerobic Earth. They also indicate a period over which the bio-availability of iron shifted from abundance to limitation, a condition that extends to the modern day. Thus, it is considered likely that the vast majority of extant organisms face the common problem of securing sufficient iron from their environment – a problem that Life on Earth has had to cope with for some 2 billion years. This struggle for iron is exemplified by the competition for this metal amongst co-habiting microorganisms who resort to stealing (pirating) each others iron supplies! The reliance of micro-organisms upon iron can be disadvantageous to them, and to our innate immune system it represents a chink in the microbial armour, offering an opportunity that can be exploited to ward off pathogenic invaders. In order to infect body tissues and cause disease, pathogens must secure all their iron from the host. To fight such infections, the host specifically withdraws available iron through the action of various iron depleting processes (e.g. the release of lactoferrin and lipocalin-2) – this represents an important strategy in our defence against disease. However, pathogens are frequently able to deploy iron acquisition systems that target host iron sources such as transferrin, lactoferrin and hemoproteins, and thus counteract the iron-withdrawal approaches of the host. Inactivation of such host-targeting iron-uptake systems often attenuates the pathogenicity of the invading microbe, illustrating the importance of ‘the battle for iron’ in the infection process. The role of iron sequestration systems in facilitating microbial infections has been a major driving force in research aimed at unravelling the complexities of microbial iron transport processes. But also, the intricacy of such systems offers a challenge that stimulates the curiosity. One such challenge is to understand how balanced levels of free iron within the cytosol are achieved in a way that avoids toxicity whilst providing sufficient levels for metabolic purposes – this is a requirement that all organisms have to meet. Although the systems involved in achieving this balance can be highly variable amongst different microorganisms, the overall strategy is common. On a coarse level, the homeostatic control of cellular iron is maintained through strict control of the uptake, storage and utilisation of available iron, and is co-ordinated by integrated iron-regulatory networks. However, much yet remains to be discovered concerning the fine details of these different iron regulatory processes. As already indicated, perhaps the most difficult task in maintaining iron homeostasis is simply the procurement of sufficient iron from external sources. The importance of this problem is demonstrated by the plethora of distinct iron transporters often found within a single bacterium, each targeting different forms (complex or redox state) of iron or a different environmental condition. Thus, microbes devote considerable cellular resource to securing iron from their surroundings, reflecting how successful acquisition of iron can be crucial in the competition for survival. The aim of this book is provide the reader with an overview of iron transport processes within a range of microorganisms and to provide an indication of how microbial iron levels are controlled. This aim is promoted through the inclusion of expert reviews on several well studied examples that illustrate the current state of play concerning our comprehension of how iron is translocated into the bacterial (or fungal) cell and how iron homeostasis is controlled within microbes. The first two chapters (1-2) consider the general properties of microbial iron-chelating compounds (known as ‘siderophores’), and the mechanisms used by bacteria to acquire haem and utilise it as an iron source. The following twelve chapters (3-14) focus on specific types of microorganism that are of key interest, covering both an array of pathogens for humans, animals and plants (e.g. species of Bordetella, Shigella, , Erwinia, Vibrio, Aeromonas, Francisella, Campylobacter and Staphylococci, and EHEC) as well as a number of prominent non-pathogens (e.g. the rhizobia, E. coli K-12, Bacteroides spp., cyanobacteria, Bacillus spp. and yeasts). The chapters relay the common themes in microbial iron uptake approaches (e.g. the use of siderophores, TonB-dependent transporters, and ABC transport systems), but also highlight many distinctions (such as use of different types iron regulator and the impact of the presence/absence of a cell wall) in the strategies employed. We hope that those both within and outside the field will find this book useful, stimulating and interesting. We intend that it will provide a source for reference that will assist relevant researchers and provide an entry point for those initiating their studies within this subject. Finally, it is important that we acknowledge and thank wholeheartedly the many contributors who have provided the 14 excellent chapters from which this book is composed. Without their considerable efforts, this book, and the understanding that it relays, would not have been possible. Simon C Andrews and Pierre Cornelis

An economic decision support tool for simulating paratuberculosis control strategies in a UK suckler beef herd

A dynamic, deterministic, economic simulation model was developed to estimate the costs and benefits of controlling Mycobacterium avium subsp. paratuberculosis (Johne's disease) in a suckler beef herd. The model is intended as a demonstration tool for veterinarians to use with farmers. The model design process involved user consultation and participation and the model is freely accessible on a dedicated website. The 'user-friendly' model interface allows the input of key assumptions and farm specific parameters enabling model simulations to be tailored to individual farm circumstances. The model simulates the effect of Johne's disease and various measures for its control in terms of herd prevalence and the shedding states of animals within the herd, the financial costs of the disease and of any control measures and the likely benefits of control of Johne's disease for the beef suckler herd over a 10-year period. The model thus helps to make more transparent the 'hidden costs' of Johne's in a herd and the likely benefits to be gained from controlling the disease. The control strategies considered within the model are 'no control', 'testing and culling of diagnosed animals', 'improving management measures' or a dual strategy of 'testing and culling in association with improving management measures'. An example 'run' of the model shows that the strategy 'improving management measures', which reduces infection routes during the early stages, results in a marked fall in herd prevalence and total costs. Testing and culling does little to reduce prevalence and does not reduce total costs over the 10-year period.

The Euro+Med treatment of Hedera (Araliaceae) - recent studies and a new name

For the treatment of the Hedera taxa present in the territories included in the Euro+Med PlantBase project a new name is required and here validated, Hedera rhizomatifera, based on Hedera helix subsp. rhizomatifera.

The uses of Atuna racemosa Raf. (Chrysobalanaceae) in Samoa

An ethnobotanical study was made of the uses of Atuna racemosa subsp. racemosa (Chrysobalanaceae) in Samoa. The main use is of the cotyledons to extract an anti-inflammatory massage oil and a putty to caulk boats. Minor uses as a medicinal and of the wood are reported and a survey of herbarium material shows that the fruit of Atuna is widely used throughout the Pacific region.

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(LyS(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Val(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC)) -tRNA(Val(UAC)) -tRNA(Ala(UGC)) and tRNA(Glu(UUC)) -tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and inter-cistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), R damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).

Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media

Aims: To make a preliminary assessment of the incidence of Salmonella in Egyptian dairy products, and to investigate the effectiveness of various protocols for the detection of the pathogen in these products. Methods and Results: Samples of milk and related dairy products were randomly collected from local markets and examined for the presence of Salmonella. While most samples were free of the organism, isolates of Salmonella enterica subsp. enterica serovar Typhimurium PT 8 could be recovered from 'matared' cream specimens. These isolates were susceptible to antibiotics usually used to challenge infections caused by Salmonella. A combination of buffered peptone water, Muller-Kauffman tetrathionate broth, and brilliant green phenol red agar gave the best results for the detection of the pathogen. Selenite-cystine broth and Hektoen enteric agar were ineffective as an enrichment and a plating medium, respectively, in the isolation of Salmonella. A modified identification strategy that reduces the burden of serological testing of presumptive isolates is proposed. Conclusions, Significance and Impact of the Study: 'Matared' cream could be a vehicle for transmitting Salmonella. Using the above combination of media, beside the suggested modified confirmatory procedure, should increase the effectiveness and ease of the detection of Salmonella in milk and dairy products.

Transfer of members of the genus Falcivibrio to the genus Mobiluncus, and emended description of the genus Mobiluncus

It has long been thought that the genera Mobiluncus and Falcivibrio contain the same organisms. Using a polyphasic approach, it was found that Mobiluncus curtisii and Mobiluncus mulieris were the same as Falcivibrio vaginalis and Falcivibrio grandis, respectively. As the genus name Mobiluncus takes precedence, it is proposed that F. vaginalis and F. grandis be transferred to the genus Mobiluncus. In agreement with previous studies, results from phenotypic tests did not support the separation of M. curtisii strains into its two subspecies, M. curtisii subsp. curtisii and M. curtisii subsp. holmesii. Phenotypic complexity within M. curtisii dictates that the species should be treated as a complex until more in-depth analyses of the species have been performed. Phylogenetic analyses, based on 16S rRNA gene sequences, demonstrated that the genus Mobiluncus was associated with Varibaculum cambriense and the two subspecies of Actinomyces neuii, and that A. neuii is only distantly related to Actinomyces sensu stricto.

Identification of Bartonella species in rodents, shrews and cats in Denmark: detection of two B-henselae variants, one in cats and the other in the long-tailed field mouse

Small mammals and stray cats were trapped in two areas of North Zealand, Denmark, and their blood cultured for hemotrophic bacteria. Bacterial isolates were recovered in pure culture and subjected to 16S rDNA gene sequencing. Bartonella species were isolated from five mammalian species: B. grahamii from Microtus agrestis (field vole) and Apodemus flavicollis (yellow-necked field mouse); B. taylorii from M. agrestis, A. flavicollis and A. sylvaticus (long-tailed field mouse); B. tribocorum from A. flavicollis; R vinsonii subsp. vinsonii from M. agrestis and A. sylvaticus; and B. birtlesii from Sorex vulgaris (common shrew). In addition, two variant types of B. henselae were identified: variant I was recovered from three specimens of A. sylvaticus, and B. henselae variant 11 from I I cats; in each case this was the only B. henselae variant found. No Bartonella species was isolated from Clethrionomys glareolus (bank vole) or Micromys minutus (harvest mouse). These results suggest that B. henselae occurs in two animal reservoirs in this region, one of variant I in A. sylvaticus, which may be transmitted between mice by the tick Ixodes ricinus, and another of variant 11 in cats, which may be transmitted by the cat flea (Ctenocephalides felis). To our knowledge, this is the first report of the occurrence of B. henselae and B. tribocorum in Apodemus mice.

Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine

Aims: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. Methods and Results: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. Conclusions: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. Significance and impact of the Study: Selected strains could be used as adjunct starters to make high quality Feta cheese.

A study of the effects of adjunct cultures on the aroma compounds of Feta-type cheese

The influence of adjunct brine cultures on the volatile compounds in Feta-type cheeses made from bovine milk was studied. Four batches of brine were produced: one with no added adjuncts, a second containing Lactobacillus paracasei subsp. paracasei, a third containing Lb. paracasei subsp. paracasei plus Debaryomyces hansenii and a fourth with Lb. paracasei subsp. paracasei plus Yarrowia lipolytica. All the cultures were isolated from commercial Feta brines. Aroma compounds were analysed by dynamic headspace analysis, on-line coupled with GC/MS. The most important volatile compounds were quantified in the experimental cheeses; it was concluded that the use of Lb. paracasei subsp. paracasei and D. hansenii as adjuncts in the manufacture of Feta-type cheeses contribute to the formation of a richer pattern of aroma compounds, namely alcohols, aldehydes and esters. The inclusion of Y. lipolytica resulted in the production of undesirable aroma compounds that are not part of the usual volatile profile of high quality Feta cheeses. (C) 2004 Elsevier Ltd. All rights reserved.

Examination of faecal Bifidobacterium populations in breast- and formula-fed infants during the first 18 months of life.

Bifidobacteria in the infant faecal microbiota have been the focus of much interest, especially during the exclusive milk-feeding period and in relation to the fortification of infant formulae to better mimic breast milk. However, longitudinal studies examining the diversity and dynamics of the Bifidobacterium population of infants are lacking, particularly in relation to the effects of weaning. Using a polyphasic strategy, the Bifidobacterium populations of breast- and formula-fed infants were examined during the first 18 months of life. Bifidobacterium-specific denaturing gradient gel electrophoresis demonstrated that breast-fed infants harboured greater diversity than formula-fed infants and the diversity of the infants' Bifidobacterium populations increased with weaning. Twenty-seven distinctive banding profiles were observed from ∼1100 infant isolates using ribosomal intergenic spacer analysis, 14 biotypes of which were confirmed to be members of the genus Bifidobacterium. Two profiles (H, Bifidobacterium longum subsp. infantis; and I, Bifidobacterium bifidum) were common culturable biotypes, seen in 9/10 infants, while profile E (Bifidobacterium breve) was common among breast-fed infants. Overall, inter- and intra-individual differences were observed in the Bifidobacterium populations of infants between 1 and 18 months of age, although weaning was associated with increased diversity of the infant Bifidobacterium populations. Breast-fed infants generally harboured a more complex Bifidobacterium microbiota than formula-fed infants.

Rice seed quality development and temperature during late development and maturation

The potential longevity of japonica rice (Oryza sativa L. subsp. japonica) seed is particularly sensitive to high temperature – and thus climate change – during development and maturation. Cultivar Taipei 309 was grown at 28/208C (12 h/12 h) and then from 19 DAA (days after 50% anthesis), when seeds were just over half filled, at 28/208C, 30/228C, 32/248C or 34/268C (12 h/12 h). Whereas ability to germinate ex planta had been achieved in almost all seeds by 24 DAA, only half the population were desiccation tolerant. Desiccation tolerance continued to increase over the subsequent 28 d, similarly at all four temperatures. Subsequent longevity, assessed by p50 (period in days to reduce viability to 50% in hermetic storage at 408C with c. 15% moisture content), increased progressively at 28/208C until 38 DAA, and remained constant until the final harvest (52 DAA). The three warmer temperature regimes provided similar longevity to 28/208C at any one harvest, except at 38 DAA where the warmest (34/268C) was poorer. That temperature regime also provided greater seed-to-seed variability within each survival curve. The results confirm that appreciable improvement in seed quality occurs during seed development and also subsequent maturation in japonica rice, but that increase in temperature from 28/208C to 34/268C during late seed filling onwards has comparatively little effect thereon. Comparison with previous investigations suggests that seed quality development may be less sensitive to high temperatures during late development and maturation than during the early seed development that precedes it.