Guttation Droplets Research Articles

Stachybotrys spp. and the guttation phenomenon

The formation of guttation droplets is a long-known property of various fungi. However, their composition, biological function and metabolism in fungi have hardly attracted deeper research interest. The highly toxic mould Stachybotrys (S.) chartarum chemotype S is supposed to play-amongst other factors such as endotoxins and microbial volatile organic compounds (MVOCs)-an important role in indoor air toxicity, mainly after water damage. The way of toxins becoming airborne and leading to exposure via inhalation, however, is still under discussion. We hypothesised that guttation may be a factor for exudation of toxins into the environment. Therefore, selected isolates (n = 15) of our own culture collection of Stachybotrys spp. (S. chartarum chemotype S, S. chartarum chemotype A, S. chlorohalonta) originating from various habitats were cultivated on malt extract agar for 3weeks. All strains but one produced different amounts of guttation droplets, which were collected quantitatively and subjected to various independent analytical techniques like ELISA, effect-based bioassay (MTT cell culture test) and tandem mass spectrometry (LC-MS/MS). Actually, the toxigenic isolates (n = 5) produced highly toxic guttation droplets, which was confirmed by all methods. The concentration of macrocyclic trichothecenes, such as satratoxin G and H, ranged between the LOD and 7,160ng/ml exudate and 280 and 4,610ng/ml as determined by LC-MS/MS, respectively. According to our knowledge, the ability of S. chartarum to produce toxic exudates is reported for the first time, which possibly plays an important role regarding its toxic potential in indoor environments.

Ingress of Salmonella enterica Typhimurium into Tomato Leaves through Hydathodes

Internal contamination of Salmonella in plants is attracting increasing attention for food safety reasons. In this study, three different tomato cultivars “Florida Lanai”, “Crown Jewel”, “Ailsa Craig” and the transgenic line Sp5 of “Ailsa Craig” were inoculated with 1 µl GFP-labeled Salmonella Typhimurium through guttation droplets at concentrations of 109 or 107 CFU/ml. Survival of Salmonella on/in tomato leaves was detected by both direct plating and enrichment methods. Salmonella cells survived best on/in the inoculated leaves of cultivar “Ailsa Craig” and decreased fastest on/in “Florida Lanai” leaves. Increased guttation in the abscisic acid over-expressing Sp5 plants may have facilitated the entrance of Salmonella into leaves and the colonization on the surface of tomato leaves. Internalization of Salmonella Typhimurium in tomato leaves through guttation drop inoculation was confirmed by confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can enter tomato leaves through hydathodes and move into the vascular system, which may result in the internal translocation of the bacteria inside plants.

Guttation droplets of the edible mushroom Suillus bovinus as a new source of natural antioxidants

Although guttation is a well-known feature of several filamentous fungi, their potential as natural antioxidant sources is unknown. This study investigates the antioxidant capacity of the exudate produced by the wild edible mushroom Suillus bovinus and the effect of the temperature and of light/darkness exposure on antioxidants synthesis. The fungus was grown on solid media at 18°C or 25°C, under light/darkness conditions. The antioxidant activity of the exudate was evaluated by using 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity (DPPH) and reducing power assays. The fungal exudate proved to have antioxidant activity, showing values of reducing power ranging from 0.450 to 0.528, and of scavenging effects on DPPH radicals from 75.68 to 82.38%. Colonies grown under continuous light at 18°C had the lowest growth rate and exudate volume, but these environmental conditions proved to be the most appropriate to increase antioxidant activity of the droplets. These results indicated that the droplets of S. bovinus are a promising source of natural antioxidants and provide useful information on how to manipulate the production of such compounds.

Assessment of risks to honey bees posed by guttation

Background: Besides their nectar and pollen collecting activities, honey bees also forage water. Guttation droplets may be used as a water source. Measurements of high residue levels of some intrinsically highly toxic, systemic insecticides in guttation droplets triggered research activities on the potential risk for honey bees. Since 2009, a large number of studies have been conducted on the environmental conditions and factors favoring guttation, foraging of guttation, the occurrence of guttation in different crops, the frequency of guttation events and residue measurements in guttation droplets in different crops, at different growth stages and with different active ingredients. Different approaches of laboratory, semi-field and field studies were set up to address the potential risk of guttation to bees and to gain clarification whether and how this concern would need to be specifically addressed in the risk assessment for bees. Results: Occasionally increased mortalities of worker bees were reported from single events in some trials, when colonies were placed directly next to the sown maize crop treated with a systemic insecticide. However, there were no long-term colony effects (e.g. on colony strength and brood development) reported from any of the realistic worst case exposure trials conducted by either public research institutes or industry. Conclusion: The potential risk for bees is in the first instance dependent on the distance of the colonies to treated crops. Maize is considered as the worst case crop in terms of frequency, duration and intensity of guttation and of residue level of compounds found in guttation liquid. Though increased worker bee mortality on individual days was seen in some of the field studies where hives were placed directly at guttating maize fields, adverse effects to colony vitality, colony and brood development were never observed. Keywords: Guttation, risk assessment, pesticides, honey bees.

Water collection by honey bees – How far will foragers fly to use water sources like guttation drops? A first distance trial using cereals and oilseed rape

Background: Depending on the location, various water sources may be available for bee colonies. These sources can be permanent, such as ponds, or incidental like dew or guttation droplets. The aim of the experiment was to investigate whether bees prefer guttation drops as a water source compared to dew or rain drops. Furthermore it was analysed if bees use these water sources up to a distance of 50m from their hives. Results: During the experiment 147 bees were observed scanning the surface of the plants without landing, 13 bees took up guttation fluid and 36 bees collected dew or rain drops. Few bees were observed collecting guttation fluid at 50m from their hives but most in close proximity of the hives. Furthermore, in some dead bees residues of the seed treatment were detected (imazalil: 0.0011 μg/bee – 0.329 μg/bee; LD50= 35.1 μg/bee). Conclusion: In the majority of observations, bees were spotted scanning the leaf area of the plants. Only single bees were observed that actually took up water from plant leaves. It seems these bees did not distinguish between dew, rain or guttation droplets. The majority of water collecting or bees resting on plants were observed in the close proximity of their hives. Keywords: guttation fluid, pesticides, seed treatment, honeybee, water source, distance

Orientating experiments on guttation fluid of seed treated maize ( Zea mays L.) in relation to the water collecting behaviour of honey bees ( Apis mellifera L.)

The major task of this study was to examine the honey bee’s (Apis mellifera L.) water collection behaviour in relation to the process of guttation in a corn plot treated with clothianidin. As shown in experiments 2009 under field conditions, guttation fluid of seed coated maize contains concentrations up to 8000 ng ml. Based on these results, further investigations were conducted to examine the honey bee’s water collection behaviour in relation to designated water collection stations and varying water quality under tent conditions. In the ‘corn tent’ a high number of maize seed coated with clothianidin (Poncho and Poncho Pro, Bayer CropScience) was sown to create an extreme situation for the bees. Beside the guttation no alternative water sources were offered. In the ‘mesh tent’ the soil was covered with texture to exclude natural water sources so defined water sources were placed at varying distances and with varying qualities of water. Pollen and sugar dough were offered on feeding stations Observations on bees drinking at the water sources, their behaviour and reaction on water quality were induced in both tents. The instances of death in the experimental bee colonies were regularly noted, and the dead bees collected. These tent experiments showed, that bees, which collected guttation droplets in seed dressed corn or clothianidin-spiked water at the artificial water source return to the colony and get damaged after a certain time with the known symptoms. Dead bees can be found in the colony as well as in front of the hive. The number of affected bees in the colony is limited but under the chosen conditions the consumption of contaminated water led to a reduced colony development.

Frequency and intensity of guttation events in different crops in Germany

Background: In crops of economic relevance and some common weeds in Germany, several glasshouse and field trials were conducted at the JKI and cooperating research partners (DWD, IfZ, BDP and UFOP) from 2009 to 2011. Aim of the investigations was to analyze the guttation frequency of these crops, to document climatic conditions that trigger guttation and to describe the quality and quantity of guttation events (e.g. size/number of drops). Results: Guttation occurred in all crops mainly at young growth stages. Only maize, cucumber, oilseed rape and potato produced guttation droplets until emergence of inflorescence. The excreted amount of fluid was comparable for maize and oilseed rape but considerably lower for sugar beet and barley. In field situation guttation occurred frequently in more than one plant species in parallel when it was not dry or too windy or frosty. Conclusion: Monocotyledonous crops and weeds showed a higher guttation frequency than most dicotyledonous crops. Maize showed large guttation drops even under low relative air humidity, whereas guttation drops in sugar beets were much smaller and only observed under very high humidity conditions (>90%). Other dicotyledonous crops, such as oilseed rape and potato, showed a much higher guttation frequency than sugar beets. Keywords: guttation, honey bees, pesticides, crops, weeds

The significance of guttation in the secondary spread of Clavibacter michiganensis subsp. michiganensis in tomato greenhouses

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), can spread in commercial tomato greenhouses causing epidemics. Results of greenhouse experiments with Cmm‐contaminated tools demonstrated disease spread for only a limited distance (<4 plants) from infected plants. However, touching symptomless infected plants bearing guttation droplets prior to touching nearby plants spread the pathogen over longer distances within rows (>22 plants). The pathogen was exuded in large numbers in the guttation fluid of infected plants; its presence in the guttation fluid was not influenced by the inoculation procedures, leaf age or the volume of the guttation droplets. Population size of Cmm and the incidence of leaflets with epiphytic bacteria were significantly higher in plants placed in a guttation‐induction chamber than in those kept in a growth chamber with high humidity, suggesting exudation through guttation contributed to the formation of epiphytic populations on leaflets. This new knowledge may provide a simple and environmentally friendly means for decreasing the spread of the disease by avoiding contact with plants during periods when they bear guttation droplets.

Mycotoxin production by different ochratoxigenic Aspergillus and Penicillium species on coffee- and wheat-based media

Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8μg/g) and by A. niger BFE632 (10.6μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media.

Influence of Leaf Tolerance Mechanisms and Rain on Boron Toxicity in Barley and Wheat

Boron (B) toxicity is common in many areas of the world. Plant tolerance to high B varies widely and has previously been attributed to reduced uptake of B, most commonly as a result of B efflux from roots. In this study, it is shown that the expression of genes encoding B efflux transporters in leaves of wheat (Triticum aestivum) and barley (Hordeum vulgare) is associated with an ability of leaf tissues to withstand higher concentrations of B. In tolerant cultivars, necrosis in leaves occurred at B concentrations more than 2-fold higher than in sensitive cultivars. It is hypothesized that this leaf tolerance is achieved via redistribution of B by efflux transporters from sensitive symplastic compartments into the leaf apoplast. Measurements of B concentrations in leaf protoplasts, and of B released following infiltration of leaves, support this hypothesis. It was also shown that under B-toxic conditions, leaching of B from leaves by rain had a strong positive effect on growth of both roots and shoots. Measurements of rates of guttation and the concentration of B in guttation droplets indicated that the impact of guttation on the alleviation of B toxicity would be small.

Guttation droplets of Penicillium nordicum and Penicillium verrucosum contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10-14 days at 25 degrees C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25 degrees C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7-8667.0 ng OTA and 159.7-2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0-819.3 ng OTA and 4.5-409.7 ng OTB/g) and fungal free agar (15.3-417.0 ng OTA and 12.7-151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.

Overexpression of GLUTAMINE DUMPER1 leads to hypersecretion of glutamine from Hydathodes of Arabidopsis leaves.

Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.

Guttation - the basis of an assay for evaluating formulation behaviourin vivo

Guttation droplets collected from the tips of winter leaves, previously treated with a 14 C version of an experimental xylem-mobile fungicide (ExpF) that was known to elute readily in guttation fluid, were analysed for the presence of radiolabel. The effects of adjuvants on the elution rate was investigated and related to the known biological profile of the fungicide when used in combination with adjuvants. This method, using ExpF as a model molecule, is undergoing further development as a means of investigating formulation behaviour in vivo. Not all xylem-mobile fungicides elute significantly and data are presented to illustrate this.

Ingress of Clavibacter michiganensis subsp. michiganensis into Tomato Leaves Through Hydathodes

ABSTRACT Hydathodes of tomato leaves served as extremely efficient infection courts for the bacterial canker pathogen, Clavibacter michiganensis subsp. michiganensis. Chlorotic lesions developed at the tips of leaflet lobes about 2 weeks after inoculation of guttation droplets. Lesions expanded along the leaflet margins and became necrotic. Movement of C. michiganensis subsp. michiganensis from the inoculated leaflet into the rachis was slow and erratic. Histological observations revealed that pathogen populations first developed within large intercellular spaces lying beneath the stomata, which serve as water pores in tomato hydathodes. Bacteria were first observed within vessels of the large marginal fimbriate veins 7 days after inoculation. By 14 days after inoculation, large populations could be seen within the vessels; and by 21 days after inoculation, tissue collapse was widespread and masses of bacteria could be seen in the intercellular spaces and within necrotic cells.

Dhurrin-6′-glucoside, a cyanogenic diglucoside from Sorghum bicolor

A novel cyanogenic diglucoside has been isolated from methanolic extracts of young seedlings of Sorghum bicolor. Its structure was established as dhurrin-6-glucoside from NMR, mass spectrometry and enzymatic hydrolysis data. Compared with dhurrin, which is the major cyanogenic glucoside in Sorghum leaves, dhurrin-6-glucoside occurs only in low concentrations. In contrast, however, the diglucoside is present in significant amounts in guttation droplets of young Sorghum seedlings. The presence of the diglucoside and its occurrence in apoplasmic exudates supports the hypothesis that diglucosides represent metabolites of cyanogenic monoglucosides which can be translocated within the plant.

Dew and guttation: formation and environmental significance

Observations were made of dew and guttation droplets on the grass Holcus lanatus L. (Yorkshire fog) at a site in rural Norfolk, UK, in 1985. The mean overnight dewfall was 0.14 ± 0.02 mm ( N = 40 nights). Guttation droplets found on grass blade tips had an average diameter of 1.49 ± 0.16 mm ( N = 1200 measurements), compared with 0.20 = 0.02 mm for true dew droplets ( N = 550 measurements). The average total volume of guttation water exuded per grass blade per night was 1.0 ± 0.3 × 10 −7 dm 3, which represents about 0.1 mm of precipitation; guttation supplied about the same amount of water as dewfall to a short grass surface. About 8% of the mean daily June–August net radiation in southern England would be needed to evaporate the average dew and guttation-derived leaf wetness, which totalled 0.25 ± 0.04 mm. Guttation amount was significantly correlated with soil temperature and moisture ( P < 0.001, r 2 = 0.874). The diameter of dew droplets was proportional to gravimetrically measured dewfall amount. The average total surface area of dewdrops on short grass was about 11 m 2 m −2 ground area. The corresponding average for guttation was about 5 m 2 m −2 ground area. The modal pH of a mixture of dewfall and guttation was 5.0–5.2. Chemical analyses of mixed samples of dew and guttation showed a ten-fold super-saturation with respect to CO 2. The equilibrium partial pressure of SO 2 in these bulked samples was very low (10 −4−10 −3 Pa), an order of magnitude lower than atmospheric concentrations. The half-life of SO 2 with respect to oxidation in the dew and guttation mixtures was a few hours.

A hydathode inoculation technique for the simulation of natural black rot infection of cabbage by Xanthomonas campestris pv. campestris

SummaryA technique was developed for the reliable inoculation of Xanthomonas campestris pv. campestris into the cabbage host, in a manner which simulates a natural process of penetration. By introducing the inoculum into guttation droplets on the leaf margin, the bacteria are taken into the plant via the hydathodes, thereby avoiding mechanical injury to the plant and the possibility of producing artefacts. This technique, while being highly efficient, possesses several advantages over commonly used methods of inoculation.

Distribution and Anatomy of Hydathodes in Asteraceae

Hydathodes have been reported as rare for the Asteraceae, but we collated published reports of guttation, water pores, and hydrathodes from ca. 80 genera of 10 tribes. Only a few incomplete anatomical descriptions were found. Leaf teeth with guttation droplets were removed for anatomical study from Ambrosia trifida, Arctium minus, Erigeron annuus, Eupatorium rugosum, Lactuca scariola, Rudbeckia laciniata, and Silphium perfoliatum. Conspicuous xylem, but very little phloem, enters each tooth. Phloem ends well before xylem termination. Xylem diverges distally into individual files of tracheary elements separated by parenchyma. Progressively toward the tooth apex, xylem parenchyma proliferates, and successive cells are at first more elongate with increasingly sinuous walls, then shorter, broader, and lobed. The epithem beyond xylem termination resembles ordinary mesophyll chlorenchyma. The bundle sheath is variably developed but always incomplete. Water pores resemble ordinary stomata but are mostly sunken, ...

Physiological changes in, and phosphate uptake by potato plants during development of, and recovery from phosphate deficiency

The development of phosphate deficiency (P‐stress) was observed in rooted sprouts of Solanum tuberosum L. cv. Desiree growing in solutions without phosphate. Shoot growth was inhibited by P‐stress within 3 to 5 days of terminating the phosphate supply, while significant effects on root growth were not recorded until 7 to 9 days. Thus, the shoot:root dry weight ratio decreased from 4.3 to 2.6 over a 10‐day period. Growth in the absence of an exogenous phosphate supply progressively diluted the phosphorus in the plant. The proportional decrease in concentration was similar in roots and shoots over a 7‐day period, even though the former were growing more quickly.The potential for phosphate uptake per unit weight of root increased rapidly during the first 3 days of P‐stress. When the plants were provided subsequently with a labelled, 1 mol m−3 phosphate solution, the absorption rate was 3 to 4‐fold greater than that of control plants which had received a continuous phosphate supply. The increased rate of uptake by P‐stressed plants was accounted for by an increase (3‐fold) in the Vmax of system 1 for phosphate transport and by a marked increase in the affinity of the system for phosphate (decrease in Km). In the early stages of P‐stress, before marked changes in growth were measured, the proportion of labelled phosphate translocated to the shoots increased slightly relative to the controls when a phosphate supply was restored. In the later stages of stress a greater proportion was retained in the root system of P‐stressed plants than in that of controls.In plants with roots divided between solutions containing or lacking a phosphate supply, the increased absorption rate was determined by the general demand for phosphate in the plant and not by the P‐status of the particular root where uptake was measured. By contrast, the poportion translocated was strongly dependent on the P‐status of the root.The restoration of a phosphate supply to P‐stressed plants was marked by a rapid increase in the P concentration in snoots and roots which returned to levels similar to unstressed controls within 24 h. The enhanced uptake rate persisted for at least 5 days, resulting in supra‐normal concentrations of P in both shoots and roots, and in the formation of extensive necrotic areas between the veins of mature leaves. Autoradiographs showed accumulations of 32P in these lesions and at the points where guttation droplets formed on leaves.

Development of Techlofthalam and Studies on Its Mode of Action

Studies were begun to determine the effectiveness of N-aryl-3, 4, 5, 6-tetrachlorophthalamic acids against bacterial leaf blight of rice, caused by Xanthomonas campestris pv. oryzae. The structure-activity relationship of many derivatives indicated that the presence of both the carboxyl radical and four chlorine atoms at phthalic acid moiety was structurally essential for effectiveness against the disease, and optimization of activity was obtained in a derivative (techlofthalam) having 2, 3-dichlorophenyl as the aryl group. Techlofthalam showed no activity in vitro against the causal bacterium, when estimated by a turbidimetric method. Soil application with techlofthalam was found to be considerably effective at 4kg per hectare both in the greenhouse and the field. With foliar application, however, extremely different efficacies were shown with two inoculation methods; excellent control against lesion development was realized by spray inoculation, but only poor control was obtained by needle prick inoculation. Intensive field evaluation proved that techlofthalam spray at 60 to 120g per hectare showed a stable and excellent effect. In order to know the mode of action of techlofthalam, the change in population and aggressiveness of the bacterium in leaves sprayed with a 100ppm solution were examined. There was a definite trend of change in bacterial populations at all spray times, 1 day before and 3 days after inoculation, and after lesion formation; the bacterial population started to decrease a few days after spraying, and then was stable at much lower levels. The bacterium isolated from a sprayed leaf had lower aggressiveness than one from an unsprayed leaf. In consideration of generation time (approximately 10 hours in the exponential growth phase) of the bacterium in rice leaf, these results suggested techlofthalam's direct inhibitory effect on the multiplication of bacterium on the leaf. Retardation of the increase in viable bacterial population was found by a static culture using Suwa's synthetic medium containing techlofthalam at 0.1ppm; this was practically the same concentration as that gaschromatographically determined in guttation droplets on rice leaves a few days after spraying. Some other results also gave support to the conclusion that techlofthalam displays bacteriostatic action in rice leaf. Techlofthalam has been practically used in Korea since 1979.