Adsorption of Agrobacterium tumefaciens to Susceptible Potato Tissues: a Physisorption Process

Adsorption of Agrobacterium tumefaciens cells to potato tuber disks reached equilibrium after coincubation for about 30 min. More than 10% of the number of bacteria bound at equilibrium were adsorbed within 30 s. Adsorption isotherms obtained at three temperatures showed that the heat of adsorption was nearly zero.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

Seasonal changes in the structure of the anoxygenic photosynthetic bacterial community in Lake Shunet,Khakassia

Seasonal studies of the anoxygenic phototrophic bacterial community of the water column of the saline eutrophic meromictic Lake Shunet (Khakassia) were performed in 2002 (June) and 2003 (February–March and August). From the redox zone down, the lake water was of dark green color. Green sulfur bacteria predominated in every season. The maximum number of green sulfur bacteria was 10⁷ cells/ml in summer and 10⁶ cells/ml in winter. A multi-syringe stratification sampler was applied for the study of the fine vertical distribution of phototrophs in August 2003; the sampling was performed every 5 cm. A 5-cm-thick pink-colored water layer inhabited by purple sulfur bacteria was shown to be located above the layer of green bacteria. The species composition and ratio of purple bacterial species depended on the sampling depth and on the season. In summer, the number of purple sulfur bacteria in the layer of pink water was 1.6 × 10⁸ cells/ml. Their number in winter was 3 × 10⁵ cells/ml. In the upper oxygen-containing layer of the chemocline the cells of purple nonsulfur bacteria were detected in summer. The maximum number of nonsulfur purple bacteria, 5 × 10² cells/ml, was recorded in August 2003. According to the results of the phylogenetic analysis of pure cultures of the isolated phototrophic bacteria, which were based on 16S rDNA sequencing, green sulfur bacteria were close to Prosthecochloris vibrioformis, purple sulfur bacteria, to Thiocapsa and Halochromatium species, and purple nonsulfur bacteria, to Rhodovulum euryhalinum and Pinkicyclus mahoneyensis.     

Antibiofilm properties of chemically synthesized silver nanoparticles found against Pseudomonas aeruginosa

Nanomedicine is now being introduced as a recent trend in the field of medicine. It has been documented that metal nanoparticles have antimicrobial effects for bacteria, fungi and viruses. Recent advances in technology has revived the use of silver nanoparticles in the medical field; treatment, diagnosis, monitoring and control of disease. It has been used since ancient times for treating wide range of illnesses. Bacterial cells adheres to surfaces and develop structures known as biofilms. These structures are natural survival strategy of the bacteria to invade the host. They are more tolerant to commonly used antimicrobial agents, thus being more difficult to be controlled. This leads to increase in severity of infection. In this study, we have investigated the effect of silver nanoparticles in the formation of biofilm in multidrug resistant strains of Pseudomonas aeruginosa. Observation showed that biofilm formation occurred at bacterial concentration of 10⁶ cfu/ml for the sensitive strain of P. aeruginosa while in the resistant strain, the biofilm was evident at bacterial concentration of about 10³ cfu/ml. The biofilm were then tested against various concentrations of silver nanoparticles to determine the inhibitory effect of the silver nanoparticles. In the sensitive strain, 20 μg/ml of silver nanoparticles inhibited the growth optimally at bacterial concentration of 10⁴ cfu/ml with an inhibition rate of 67%. Similarly, silver nanoparticles inhibited the formation of biofilm in the resistant strain at an optimal bacterial concentration of 10⁵ cfu/ml with an inhibition rate of 56%. Thus, silver nanoparticles could be used as a potential alternative therapy to reduce severity of disease due to P. aeruginosa infections.     

Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots

The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 10⁵ CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 10⁸ CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.     

Selective binding of polymyxin B to negatively charged lipid monolayers

It is demonstrated by direct measurement of surface radioactivity that the cationic polypeptide antibiotic polymyxin B is specifically adsorbed to negatively charged lipid monolayers. The latter attracted the following amounts of the biologically active $\text{mono}\text{-N[}{}^{14}\text{C]}\text{acetylpolymyxin B}$ derivative (PX): lipid A from Proteus mirabilis, 0.17; phosphatidic acid, 0.12; phosphatidylglycerol and phosphatidylserine, 0.11; dicetylphosphate, 0.107; sulfoquinovosyldiglyceride, 0.104; phosphatidylinositol and cardiolipin, 0.095; and phosphatidylethanolamine, 0.017 μg/cm². Adsorption of PX to phosphatidylcholine, monogalactosyldiglyceride and stearylamine was almost or completely zero. Total lipids from Escherichia coli adsorbed 0.057 in comparison to 0.051 μg PX/cm² of an artificial mixture of phosphatidylethanolamine/phosphatidylglycerol/cardiolipin in the proportions 75 : 25 : 5. The concentration of the surface active PX at the air/water interphase was 0.091 μg/cm². These saturation surface concentrations of PXat lipid monolayers were reached at 1 μg/ml bulk concentrations in 2 mM NaCl/1 mM Tris · HCl, pH 7.2. They decreased with decreasing surface charge density of the adsorbing monolayer. In an experiment with cardiolipin/phosphatidylethanolamine mixtures it was shown that two molecules of cardiolipin induced adsorption of one molecule PX giving a 1 : 1 ratio with regard to positive and negative charges. This could be due to a similar charge density of about one charge per 40–50 Ų in PX and lipid bilayers composed of phospholipids. The electrostatic PX-lipid interaction was severely inhibited by 10^?2 and 10^?1 M Ca²⁺ and Na⁺, respectively. It is discussed that the specificity of PX against Gram-negative bacteria is caused by the occurrence of lipid A, phosphatidylglycerol and cardiolipin at the cell surface of these microorganisms.     

Relationship between Rapid, Firm Adhesion and Long-Term Colonization of Roots by Bacteria

For rhizobacteria to exert physiological effects on plant growth, the bacteria must first effectively colonize the root surface. To examine the relationship between long-term colonization of root systems and adherence to roots in the short term, a binding assay was developed. Adherence was determined by incubating roots of intact radish seedlings with bacteria, washing and homogenizing the roots, and dilution plating the resulting homogenate. Irreversible binding of bacteria was rapid, reaching half-maximum by 5 min. All of the rhizosphere bacteria tested showed similar, concentration-dependent binding (ranging from 10⁴ to 10⁸ CFU/ml), as well as long-term colonization of radish roots under sterile conditions. Escherichia coli, which is not a root colonizer, showed about 10-fold less binding, but still demonstrated concentration-dependent binding and rapid kinetics of adherence at high concentrations (10⁶ to 10⁸ CFU/ml). The bacteria tested were very different with respect to source or habitat and plant response, yet they showed similar concentration-dependent binding. There was no correlation between the relative hydrophobicities of the cell surfaces of strains and the adherence of the strains to roots. Binding of Pseudomonas fluorescens E6-22 was promoted by divalent cations (Ca²⁺ and Mg²⁺) at concentrations of 5 to 10 mM, whereas monovalent cations (Na⁺ and K⁺) had little effect; electrostatic phenomena may partially explain adherence in the short term, an important prelude to long-term colonization of root surfaces.     

Genotypic differences in the uptake and distribution of radiocaesium in plants

The ability of endophytic bacteria to influence Erwinia carotovora var. atroseptica (Eca) growth and disease development was examined in potatoes. Bacterial populations isolated from within the tubers of five potato (Solanum tuberosum L.) cultivars (Kennebec, Butte, Green Mountain, Russet Burbank and Sebago) showed antibiosis toward Eca in an in vitro assay. Sebago was host to the highest percentage of bacterial isolates inhibiting Eca growth in vitro (49.5%), followed by Green Mountain (33.3%), Kennebec (29.3%), Russet Burbank (12.9%) and Butte (1.8%). Of these, Curtobacterium luteum was the most common species. Few endophytic bacteria from Butte were inhibitory to Erwinia; all were from Pantoea agglomerans. Significantly higher populations of Erwinia-inhibiting bacteria were recovered from Kennebec (1.89 × 10⁶ cfu fresh weight tuber tissue) as compared to the other cultivars; the lowest populations were recovered from Butte (0.01 × 10⁶ cfu per g fresh weight tuber tissue). Published levels of cultivar disease resistance to blackleg did not correspond to actual bacterial soft rot development (induced by Eca) in an in vivo (tuber) assay. However, bacterial soft rot development was negatively correlated with the density of tuber populations of endophytic bacteria found able to inhibit Eca growth in vitro (R=−0.879, p=0.05).     

Heterotrophic bacteria attached to seaweeds

Viable counts of heterotrophic bacteria attached to the green algae, Monostroma nitidum Wittrock and Enteromorpha linza (Linné) J. Agardh, ranged from 10⁴ to 10⁶/cm², and those attached to the red alga Porphyra suborbiculata Kjellman from 10³ to 10⁴/cm². These bacterial populations were larger than those attached to the brown alga Eisenia bicyclis (Kjellman) setchell ranging from 10¹ to 10⁴/cm². The bacterial populations in the environmental sea water. Nabem Inlet and Otsuchi Bay (Japan), were 10³/ml. Orange and yellow pigmented bacteria were predominant on the green and red algae, but not in the bacterial populations of the brown alga and the sea water. Most of the pigmented bacteria were identified as belonging to the Flavobacterium-Cytophaga group. A beneficial relationship was suggested between the green algae and the pigmented bacteria. Proportions of Vibrionaceae were small on the green algae.     

Effects of Inorganic Particles on Metabolism by a Periphytic Marine Bacterium

Measurements were made of adsorption of a periphytic marine bacterium, glucose, and glutamic acid to inorganic particles in seawater and defined bacterial growth medium. Measurements of the metabolism of bacteria were made in the presence and absence of particles by microcalorimetry and radiorespirometry. It was found that hydroxyapatite adsorbs glutamic acid, but not glucose, from the experimental medium. It was also found that hydroxyapatite adsorbs essentially all of the bacteria from the medium when the bacterial concentration is approximately 6 × 10⁵ bacteria per ml. If the bacterial concentration is approximately 6 × 10⁷, then only a small fraction of cells become attached. It was therefore possible to select bacterial concentrations and organic nutrients so that bacterial attachment, organic nutrient adsorption, or both would occur in different experiments. In this experimental system the metabolism by attached and nonattached bacteria of adsorbing and nonadsorbing organic nutrients was measured. The results show that bacterial activity in this model system was not enhanced by the particles, regardless of whether the bacteria, the organic nutrient, or both were associated with the surface. In fact, the respiratory activity of the attached bacteria was diminished in comparison with that of free bacteria.     

Porphyromonas gingivalis with either Tannerella forsythia or Treponema denticola induces synergistic IL-6 production by murine macrophage-like J774.1 cells

BackgroundChronic periodontitis is caused by mixed bacterial infection. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are frequently detected in deep periodontal pockets. We demonstrate that these bacteria induce proinflammatory cytokine production by the mouse macrophage-like cell line J774.1.Materials and methodsJ774.1 cells were incubated with and without bacteria for 24 h in 96-well flat-bottomed plates. The culture supernatants were analyzed by enzyme-linked immunosorbent assay for secreted mouse interleukin (IL)-6, monocyte chemoattractant protein-1, IL-23, IL-1β and tumor necrosis factor-α. The cytokine concentrations were determined using a standard curve prepared for each assay.ResultsMixed infection with P. gingivalis and either T. forsythia or T. denticola at 10⁵ CFU/ml acted synergistically to increase IL-6 production, but not monocyte chemoattractant protein-1, IL-23, IL-1β or tumor necrosis factor-α production. Gingipain inhibitors KYT-1 and KYT-36 inhibited IL-6 production by J774.1 cells incubated with 10⁵ CFU/ml of mixed bacteria.ConclusionThese results suggest that P. gingivalis with either T. forsythia or T. denticola directly induces synergistic IL-6 protein production and that gingipains play a role in this synergistic effect.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments

The movement of bacteria through groundwater is a poorly understood process. Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow. The identification of transported bacteria is often a limiting factor in both laboratory and field transport experiments. Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harboring the pGFP plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1. The Tn5GFP1 transposon incorporates the gene (gfp) encoding green fluorescent protein (GFP) and can be used to mutagenize Gram^- bacteria. Fluorescent colonies were suspended in phosphate-buffered saline (PBS) at a concentration of approx. 10⁹ bacteria/ml. A 10-cm glass column packed with quartz sand (diameter range 177–250 μm) was equilibrated with PBS prior to the forced flow introduction of the bacteria. Collected fractions were analyzed and the bacteria quantitated using a fluorescence spectrometer. Results demonstrate that the bacteria can be accurately tracked using their fluorescence, and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria. The data show a rapid breakthrough of the bacteria followed by a characteristic curve pattern. A lower limit of detection of 10⁵ cells was estimated based on these experiments. The Tn5GFP1 transposon should become a valuable tool for labeling bacteria.     

Interaction of Escherichia coli B and B/4 and Bacteriophage T4D with Berea Sandstone Rock in Relation to Enhanced Oil Recovery

Much research and development is needed to recover oil reserves presently unattainable, and microbially enhanced oil recovery is a technology that may be used for this purpose. To address the problem of bacterial contamination in an oil field injection well region, we connected each end of a Teflon-sleeved Berea sandstone rock to a flask containing nutrient medium. By inoculating one flask with Escherichia coli B, we could observe bacterial growth in the uninoculated flask resulting from the transport and establishment of cells across the rock. Differences in bacterial populations occurred depending on whether bacteriophage T4D was first adsorbed to the rock. The results of these experiments indicate that the inhibition of bacterial establishment within a rock matrix is possible via lytic interaction. Some nonlytic effects are also implied by experiments with B/4 cells, which are T4D-resistant mutants of E. coli B. A 10 to 40% retention of T4 by the rock occurred when it was loaded with 10⁵ to 10⁶ PFU. We also describe a lysogenic system for possible use in microbially enhanced oil recovery techniques.     

Insecticidal activity ofBacillus thuringiensis strains against the egyptian cotton leaf wormSpodoptera littoralis [Lep.: Nocutidae]

Among more than 50 isolates ofBacillus thuringiensis Berliner (B.t.) tested, 7 incited 100% mortality when 2nd instar larvae ofSpodoptera littoralis Boisduval were fed on alfalfa leaves dipped in a spore-crystal suspension of 10⁸ colony forming units/ml. Among those isolates,B.t. 24 demonstrated the highest activity. Larvae of instars 1 and 2 were the most susceptible toB.t. Susceptibility decreased with larval development. However, larvae of all instars were killed by isolateB.t. 24. Larvae that survived after feeding withB.t. 24 were retarded and fed less. Their weight relative to the controls was lower as the spore concentration on the leaves on which they fed was higher. Survival of the spores in the field dropped drastically to 2% after 4 days. Insecticidal activity of the sprayed suspension on those leaves, however, remained significant.B.t. 24 was also effective against larvae on cotton plants in the greenhouse and in a preliminary field experiment. Numbers of colony forming units recovered from leaves dipped in suspension of various spore concentrations showed significant correlation with the initial concentrations as did sprayed leaves. However, colony forming units recovered from sprayed leaves were 5–7.5 fold lower than from dipped leaves. Dipped cotton leaves showed 3.1×10^?5 ml attached to 1 mm² leaf surface whereas sprayed ones had 6×10^?6 ml. Those data are important for the determination of spore concentrations in suspensions required for spraying. The isolateB.t. 24 was serotyped byH. de Barjac as H-6B. thuringiensis entomocidus.     

Inhibition of porcine detrusor contractility by the flavonoid fraction of Bryophyllum pinnatum – a potential phytotherapeutic drug for the treatment of the overactive bladder syndrome

Aims: To determine if the phytotherapeutic agent, Bryophyllum pinnatum, could serve as an alternative drug for the overactive bladder syndrome, and to characterise the fraction responsible for the inhibition of detrusor contractility.Methods: Fractions were prepared from the MeOH extract of B. pinnatum and further analysed by HPLC-PDA-MS. Detrusor muscle strips were prepared from porcine bladders and the electrically induced muscle contractility measured by organ bath. The effect of B. pinnatum leaf press juice (2.5–10%), a flavonoid fraction (0.1–1 mg/ml), and a bufadienolide fraction (0.1–40 μg/ml) on detrusor contractility was assessed and compared with controls (polar fraction (0.5–5 mg/ml) and oxybutynin (10⁻⁸–10⁻⁶ M)).Results: The press juice, at a concentration of 10% led to a reduction of detrusor contractility. Bladder strips treated with the flavonoid fraction showed a significant reduction of the contractility to 21.3 ± 5.2% (1 mg/ml) while the bufadienolide fraction had no inhibitory effect in the investigated concentrations. The polar fraction showed a reduction of the contractility in a pH-dependent fashion. At 10⁻⁶ M concentration oxybutynin reduced the detrusor contractility to 21.9 ± 4.7%.Conclusions: The flavonoid fraction of Bryophyllum pinnatum reduces the porcine detrusor contractility in a dose- and time-dependent manner. Fractions from B. pinnatum may be a new pharmacological approach for the treatment of OAB.     

Host-Symbiont Specificity Expressed during Early Adsorption of Rhizobium meliloti to the Root Surface of Alfalfa

Early (4 h) adsorption of Rhizobium meliloti L5-30 in low numbers to alfalfa roots in mineral solution was examined for competition with other bacterial strains. All tested competitor strains decreased the adsorption of L5-30 by extents which depended on the strain and its concentration. The decrease of adsorption by R. meliloti competitors (all of them infective in alfalfa) was nearly complete at saturation (97 to 99% decrease). All other heterologous rhizobia and Agrobacterium tumefaciens at saturating concentrations (10⁶ to 10⁷ per ml) decreased adsorption of L5-30 only partially, less than 60%. The differential effects of homologous and heterologous competitors indicate that initial adsorption of R. meliloti to the root surface of its host occurs in symbiont-specific as well as nonspecific modes and suggest the existence of binding sites on roots which are highly selective for the specific microsymbiont in the presence of other heterologous bacteria even in very unfavorable (less than 10⁻⁴) symbiont-competitor concentration ratios.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Impact of Mount St. Helens Eruption on Bacteriology of Lakes in the Blast Zone

Lakes lying within the blast zone of Mount St. Helens showed dramatic increases in heterotrophic bacterial numbers after the eruption of 18 May 1980. The total microscopic counts of bacteria in some of the most severely affected lakes were more than 10⁷ cells per ml, an order of magnitude above the counts in outlying control lakes. Likewise, the numbers of viable bacteria reached levels of more than 10⁶ cells per ml, compated with fewer than 10⁴ cells per ml in control lakes. The CPS medium used for enumeration provided growth of up to 81.5% of the bacteria during sampling of one of the blast zone lakes. The high numbers of bacteria and the efficacy of the viable enumeration procedure are evidence that the lakes have been transformed rapidly from oligotrophy to eutrophy due to the eruption and its aftermath. Organic material leached from the devastated forest vegetation is thought to be responsible for the enrichment of heterotrophs. Total coliform bacteria were found in all of the blast zone lakes, and some lakes contained fecal coliform bacteria. Klebsiella pneumoniae was the predominant total coliform and was also identified as one of the fecal coliform bacteria, although Escherichia coli was the predominant species in that category. Our data indicate that bacterial populations peaked in the outer blast zone lakes in the summer of 1980 and in most of the inner lakes during the summer of 1981.