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Deliverables
CAYACEA has carried out a system for evaluating the fruit health conditions aimed at monitoring fungi infectious diseases in fruits.
Firstly, for the implementation of this system, an infectious procedure with Botritis cinerea, which has been supplied by partner 2 (ENEA) has been used. This system has been used in collaboration with partner 1 (Multidisciplinary Institute) to monitor the resveratrol in grapes relating to the fruit health condition mentioned beforehand.
Secondly, our company has collaborated with partner 1 (Multidisciplinary Institute) and partner 3 (Department of Molecular and Laser Physics, University of Nymegen) in validating the new analysis techniques: Laser desortion coupled with resonance enhanced multiphoton ionization and time-of-flight mass spectrometry (LD + REMPI + TOFMS) and laser- based photoacoustic (PA), which have been developed and implemented by each of the partners.
This system of validation has consisted of validation procedures by partner 6 (CAYACEA) by which a series of tests have been done to set the typical parameters defining the analytical method: Linearity, selectivity, specificity, precision, accuracy, detection limit and quantification limit.
At the same time this validation has been implemented under the guidelines of the EN- ISO- 17025 Norm and has let to verify or tests the applicability of the new technologies as analysis techniques in food control.
Finally, the efficiency of the new system of storage, which has been developed by P1 has been assessed. For this assessment process, a comparative study of the Biochemical and sensory features of the treated and non - treated fruit has been conducted.
The study out comes shown that the new system of fruit storage developed by P1 do not modify the biochemical and sensory features of the fruit (at all).
A novel technique based on the combination of LD with REMPITOFMS has been developed and validated for non-volatile compound analysis in fruits. In particular, the great sensitivity and resolution, based on selective laser ionisation and mass spectrometry, as well as the possibility of direct analysis in fruit samples are the main advantages of this method.
In particular, it has been demonstrated that laser desorption is a convenient method to solve one of the long-standing needs in non-volatile analysis: that is direct sample preparation. Furthermore the applicability of the technique to thermally labile compounds, as is the case of resveratrol, has eliminated an important limitation in analytical chemistry making the technique very promising in food analysis.
The developed technique has been validated for non-volatile molecules, specifically for resveratrol. This has been accomplished following the Directives and Guidelines from the EU stipulating that the control testing and batch release of foodstuffs analysis within Europe should be performed under a quality assurance system that must progressively converge toward the European Standard EN-ISO-17025.
The results obtained are the following:
Linearity: - 0-50mg
Repeatibility: RSD < 5%
Reproducibility: RSD < 5%
Accuracy: 96%
Detection Limit: < 100pg
Quantification Limit: < 0.3ng
Several methods have been tested aiming to enhance the endogenous content of resveratrol in grapes. First investigations have been conducted on the post-harvest elicitation of this compound in grapes upon Botrytis cinerea infection. The results obtained have been displayed in Figure 4, where a significant increase in the resveratrol content is observed subsequently to the infection with a 10-fold enhancement of resveratrol by the second day after the infection (with respect to the mock-infected group) followed by a rapid decrease leading to the disappearance of the compound by the fifth day after infection. This evolution is consistent with previous in vitro investigations on the induction of resveratrol by Botrytis cinerea in leaves in which the maximum yield of resveratrol was found at the third day after infection.
Further investigations were carried out by injecting the grapes with autoclaved Botrytis, which only produced a gentle elicitation of the resveratrol content. Nevertheless, it is interesting to notice that after the infection with dead botrytis the decrease in this enhanced resveratrol content is smoother than in the previous case allowing a longer persistence.
A series of experiments has been also carried out aiming at to increase the endogenous content of resveratrol in grapes by anoxic treatments. After anoxia, an important increase of the endogenous resveratrol content in the grapes was found for treatments up to 24 hours: ca. 3-fold enhancement after 24 h of anoxia; but for longer treatments only a slight enhancement is obtained. Moreover after these long treatments, the grapes appeared damaged, especially concerning their sensory properties (colour, texture, etc.) Regarding the evolution of the resveratrol after the treatment, the results shown preference for shorter treatments: the decrease of the resveratrol in the grapes after long treatments (e.g. 24 and 48 hours) is quite rapid, being undetectable 2 days after the treatment. After short periods of anoxia the level reached is longer maintained and the decrease is smoother for shorter treatments.
We have found that anoxia suppressed Botrytis growth in vitro, but upon transfer to normal atmosphere, the fungus grew almost normally. A significant inhibition in decay development and delay in rot appearance was observed only when anoxia was applied 24 hours after inoculation, while in all other treatments, rot became evidence 48 hours following inoculation. The increase in CO2 and in ethylene in all treatments was associated with rot development.
However an inhibition of 48 hours in CO2 and ethylene emission was observed in fruit kept under anoxia 24 hours after inoculation and the amount of CO2 and ethylene accumulated in this treatment was significantly lower compared with all other treatments. The increase in ethanol which was measured during anoxia treatments was due to the changeover to anaerobic respiration which occurred during the treatment. However, a post-anoxia AA peak was observed in all anoxia-treated fruit 24 hours after transfer to normal air. The amount of both AA and ethanol gradually decreased within 48 hours following the anoxia treatments.
No significant differences were observed between control fruit and anoxia treated fruit for 24 hours in all quality traits. However, the quality of fruit treated with N2 for 48 hours was significantly lower in most of the quality traits, except in firmness, TSS and acidity, compared with the other two treatments. Anoxia treatment for 48 hours significantly inhibited colour development and 10% of the fruit suffered from severe nitrogen damage.
The overall taste of control fruit was slightly, but not significant better than anoxia treated fruit for 24 hours. Anoxia treated fruit for 48 hours had a significant bad and off flavour taste compared with the other two treatments. Forty percent of the taste panel preferred both control and 24 hour anoxia treated fruits, while only 20% preferred fruit treated with anoxia for 48 hour.
In conclusion, pre-storage N2 treatment for 24 hour seems to be the most effective to reduce and delay decay development and inhibit fruit ripening with affecting fruit quality traits and overall taste. Therefore, this non-chemical and cheap treatment seems to be obvious direction for future application, especially in developing countries where refrigeration is inadequate or during commercial distribution.
The exogenous application of resveratrol (in lab and open field experiments) proved to be a useful method to maintain and improve the natural resistance of several fruits. Experiments were carried out on grapes, and ten days after treatment, significant differences were observed between treated and non treated bunches: while the resveratrol treated bunches still maintained a physical aspect with no signal of losses or deterioration, the untreated ones were not only dehydrated but clearly infected and deteriorated with local development of fungi, as it can be expected after that period of time. The experiment has been repeated up to five times following the same procedure with similar results. In all cases, the untreated grapes were already infected and deteriorated after 5-7 days of storage at room temperature while the grapes treated with resveratrol maintained during twice this time (i.e.: 10-15 days) a physical aspect with no external signs of deterioration.
This interesting result opened the way to subsequent investigations on the effect of the trans-resveratrol exogenous application on other fruits, to demonstrate the capabilities of resveratrol as natural pesticide. The results were similar to those obtained with grapes, except for the decay time (ca. 15 for avocado pears, 30 days for tomatoes, 75 days for apples) but in all cases, the non-treated samples presented clear decay signals, dehydration and development of fungi, while the treated ones still maintained a physical aspect with no signal of losses or deterioration.
The exogenous treatment of grapes with the ethanolic extract obtained from vine leaves is much more effective than the solution of pure resveratrol probably due to other natural compounds present in the extract.
We developed laser-based photo-acoustic (PA) systems and validated them, in collaboration with P6 (Cayacea), to monitor (on-line) the volatile compounds produced by fruits during infection with B. cinerea (ethylene, ethane, acetaldehyde, ethanol, hexanal, trans-2-hexenal and cis-3-hexenol). We correlated the production of these gases with the health status of the fruits. Moreover, in order to investigate the full range of the fruit-emitted volatiles under the pathogen attack, we built a novel system based on proton-transfer reaction mass spectrometry (PTR-MS) that is complementary to the PA instrument.
The sensitive detection of these natural fruit stress-molecules allowed us to identify those compounds which can be used as early indicators for infection and/or to enhance the fruit resistance against B. cinerea. Our results, together with the additional physiological data supplied by P2 (ENEA), P6 (Cayacea) and P5 (Bern) provide a complete representation of the dynamics of pathogen infection in vitro and in stored fruits. The plant hormone ethylene was proven to be a marker for early infection. We investigated the relationship between ethylene released by the fungus in vitro and the enhanced ethylene production in B. cinerea infected tomatoes with respect to disease development.
To produce a comprehensive description of the mode of ethylene formation and action in B. cinerea, both in vitro and in vivo we used (in collaboration with P2) an effective broad range of well characterised biochemicals related to ethylene biosynthesis (Cristescu et al. "Ethylene production by Botrytis cinerea in vitro and in tomatoes", Appl. and Envir. Microbiol. 68 (11) 2002). This may lead to future applications in the post-harvest technologies based on alternative strategies for plant fresh produce protection, mainly focused on action on the fungus rather that on inhibition plant-produced ethylene, that usually associate enhanced shelf life with decreased flavour and quality.
In addition to trans-resveratrol, the ability of other natural anti-fungal compounds to inhibit Botrytis infection has been evaluated and different methods and treatments were tested in order to enhance the endogenous concentrations of these compounds in fruits. In this direction, we investigated the application of an anoxic treatment (fruits were flushed continuously with nitrogen) for a certain period by measuring ethylene emission from infected fruits. The results of our experiments demonstrated that anoxia treatment applied immediately after inoculation delayed the disease development, but did not stop it. Application of the anoxic treatment after infecting the fruit is more efficient than applied before infection. A delay in ethylene emission correlated with a delay in disease development has been also observed when natural fruit-signalling volatiles such as six-and nine-carbon aldehydes (e.g. hexanal t-2 hexenal at ratios of 1:5 and 1:10 in 1,2 propanediol, respectively) were exogenously applied to infected fruits by fumigation for 24 hours.
Based on the strong evidence concerning the anti-fungal properties of the natural volatiles emitted by fruits under pathogen attack- acetaldehyde, transgenic tomato plants that display enhanced production of this compound have been raised.
In collaboration with Univ. of Bern who generated and characterised transgenic PDC tomato plants, two lines were selected for further studies to describe acetaldehyde and ethanol evolution in fruit by mean of photo-acoustic spectroscopy and to evaluate acquired fungal resistance.
Measuring the acetaldehyde and ethanol emission from infected PDC 57A tomatoes by photo-acoustic technique showed that this transgenic line presents a better ability to produce ethanol (ethanol release higher than the wild type) and a more rapid conversion of acetaldehyde. Due to the fact that acetaldehyde is an intermediate product, its decrease in the case of transgenic line compared with the wild type can be explained by considering the possibility that PDC 57A fruits started to use immediately and more efficient this component for other processes occurring during the interaction with the pathogen, e.g. the defence against the fungus.
Finally, our results indicated that PDC gene expression results in fruit defence to pathogenesis in transgenic plants. Although no statistical evidence is provided, the data suggests that acetaldehyde is a potential antibiotic for improvement of plant resistance to pathogenesis.
The developed novel REMPI method has been applied to the analysis of natural pesticides content in different plants: namely to the investigation of natural sources of resveratrol. A correlation between the resveratrol content and the health status has been demonstrated in botrytis infected grapes: the higher the content of resveratrol the lower development of the fungus and vice versa.
Three batches of grape samples were monitored for their resveratrol content: not infected, mock-infected and botrytis infected ones. While the non-infected grapes show a constant resveratrol content through the experiment, in the mock-infected ones a sudden decrease is observed on the first day after the buffer inoculation with a smooth diminution during the subsequent days. For the botrytis infected group a significant increase in the resveratrol content is observed subsequently to the infection with a 10-fold enhancement of resveratrol by the second day after the infection; afterwards the resveratrol shows a rapid decrease leading to the disappearance of the compound by the fifth day after infection. Previous studies attributed this decrease to the oxidative dimerization of the compound by the Botrytis cinerea.
These results on the evolution of the resveratrol content in the infected grapes have been compared with the development of the Botrytis Cinerea obtained by microbiological analysis. A clear correlation between the resveratrol content and the health status of the grapes as the mycelial grown of the Botrytis appears clearly related with the resveratrol content in the grapes. Subsequently to the infection and during the first 48 hours, there is a significant production of resveratrol in the grape as a result of its defence mechanism: the fungi act as elicitor of the resveratrol content and during this period the mycelial grown is negligible. Afterwards the resveratrol content shows a rapid decreasing attributed to the oxidative dimerization of the compound by the fungus, and it is at this stage when the botrytis grown starts to be important showing an inverse dependence with the resveratrol content, specially on the fifth day, when the resveratrol content in the grapes is already negligible.
It is then clear that any method, either endogenous or exogenous, aiming at to produce an increasing of the resveratrol content in the grapes, will unambiguously lead to maintain the health status of the fruit and to decrease its spoilage.
The effectiveness of a short pre-storage hot water rinsing and brushing was tested on decay incidence, quality parameters respiration rate and ethylene evolution of pink tomato fruits that were kept for 14 days at 12°C plus 3 days at 20°C (storage and marketing simulation).
Rinsing fresh harvested tomato at 52°C for 15 seconds while brushing significantly reduced decay development after storage and marketing simulation. This pre-storage heat treatment maintained better fruit quality as evaluated by firmness, total soluble solids, acidity, and delayed fruit ripening as measured by significantly slower colour development. In addition, the respiration rate and ethylene evolution of hot water rinse and brushing at 52°C treated fruit was significantly lower than that of untreated fruit during storage and marketing simulation.
Scanning electron microscopy showed that this method removed dirt and even fungal spores from the fruit skin. Hot water rinse and brushing at 52°C for 15 seconds, or dipping the fruit in 52°C for 1 min (Hot water dip; HWD), significantly reduced decay development and completely inhibited CI symptoms after storage and marketing simulation. The hot water rinse and brushing at 52°C treatment enhanced fruit resistance against Botrytis cinerea when fruit were inoculated 24 hours after treatment. Resistance was less pronounced when the fruits were inoculated 6 hours after treatment, and disappeared completely in fruit inoculated 48 hours after hot water rinse and brushing treatment.
On a commercial scale, treatments with hot water dips may be difficult to accomplish since a 60 second treatment is considered a long treatment time. Therefore, the alternative method of a very short (15 seconds) hot water rinse and brushing and 52°C treatment would be desirable for treating freshly harvested tomatoes. This treatment extends storability up to 3 weeks at 5°C by minimizing CI and enhancing resistance against pathogens during storage.
This technology is widely used in Israel on several fresh harvested commodities. Efforts are made to expand the use of this technology on several more commodities for export and local markets. In addition, experiments are conducted to apply this technology in the food and juice industries.
The goals of this project were to evaluate the health status of inoculated tomato after harvest by several gases emissions in order to identify early infection before disease became evident. Another goal was to evaluate natural biocides, such as acetaldehyde, by anoxia treatment and to develop semi-commercial treatment to improve the fruit quality.
The production of acetaldehyde, ethanol, ethylene and carbon dioxide by ripening tomatoes at 22°C was measured following inoculation with spore suspension of Botrytis cinerea. The aim was to determine whether infections could be detected before the disease symptoms became evident. Decay became visible between day 2 and day 3 following inoculation.
However, ethylene could be detected more than 24 hours before the first decay symptom was visible. A marked increased in CO2 could be detected only at day 4 from inoculation and was associated with decay development. Acetaldehyde evolution in Botrytis-inoculated fruit increased from day 3 until day 5 from inoculation and then declined, while a marked increase in ethanol evolution was detected from day 5 only.
Ethylene might be used as an early marker for infection in harvested fresh produce. Efforts are done to develop an electronic nose to detect specific volatiles that are emitted from infected fruit. There are several devices in the market and research is conducted to fit those devices for the use of fresh harvested crop.
The laser-based photo-acoustic (PA) technique has been developed to monitor the volatiles organic compounds, such as ethylene, ethane, acetaldehyde, ethanol, hexanal, trans-2-hexenal and cis-3-hexenol in fruits.
The PA detection system consists of a line-tuneable laser (CO2 or CO) and a photo-acoustic cell, in which the gas is detected. The laser-based detector is able to distinguish between different gases by making use of their wavelength dependent fingerprint absorption. Trace gases released by the biological samples-plated fungi or infected fruits- (up to 6 per experiment) were transported to the PA cell alternately, without accumulation, through a flow system which uses air as carrier gas. Using a CO2 laser based detector, very low amounts of ethylene can be measured on-line with a detection limit of 10ppt, which makes the photo-acoustic method 3 orders of magnitude more sensitive than traditional gas chromatography analysis.
With CO laser-based detectors, acetaldehyde (detection limit 0.1 ppb) and ethanol (detection limit of 3ppb) from three samples can be analysed simultaneously.
For the detection of hexanal, trans-2-hexenal and cis-3-hexenol released by tomatoes, we have determined their absorption spectra in the CO laser wavelength regions. In the range of 1300-2000cm(-1), the acetaldehyde absorption spectrum strongly overlaps the above-mentioned gases, while in the range of 2650-3100cm(-1), hexanal, trans-2-hexenal and cis-3-hexenol spectra have shown overlap with each other. A careful study of all the absorption lines separately has been performed in order to select the right set of laser lines suited to discriminate between the different gases.
The laser-based PA detector, as well as the electric three way valves are operated fully automatically by computer programme and it can be used to perform continuous measurements for periods up to several weeks.
Non-climacteric fruits, grapes release ethylene at very low production rate almost undetectable with standard procedures. Ethylene released by non-infected and Botrytis infected grapes, with and without exogenous trans-resveratrol application was on-line monitored by LPAS. The evolution of ethylene emission from non-infected and mock-infected grapes was compared to that from grapes treated with trans-resveratrol. The laser-photoacoustic measurements showed that exogenous application of trans-resveratrol caused a decrease of ethylene emission of at least 3 times for both not infected and mock-infected fruits. Its inhibitory effect on ethylene production became evident after 10-12 h from the application. trans-Resveratrol had a significant effect also on the ethylene released by the Botrytis infected grapes. There are two aspects that have to be considered here: firstly, the trans-resveratrol treatment determined a delay of increasing ethylene emission of about two days; and secondly, the enhanced formation of ethylene for the treated grapes is 2 times less and presented a slower rate than that corresponding to the untreated ones. Moreover, we found a strong inverse relationship between ethylene production by grapes and trans-resveratrol content measured by LD + REMPI-TOFMS. The trans-resveratrol content from the non-infected fruits was higher than that corresponding to the mock-infected which drastically decreased to zero during the first day. In correlation, ethylene released by mock-infected grapes increased in the first day up to a certain level and showed higher values compared to the non-infected. For the Botrytis infected fruits, ethylene emission rises up after 48 h when the analogous content of trans-resveratrol started to decrease irreversibly.
We studied the mechanism of fruit spoilage derived from Botrytis cinerea (Botrytis) mould in tomato berry fruit. We examined growth habits, biochemistry and infectivity of five isolates of Botrytis cinerea (ref. 1) and we tested their infectivity on tomato fruits by RT-PCR approach, to monitor fruit (tomato) and fungus (Botrytis) gene expression (ref. 2).
Our analysis based on the monitoring of specific metabolic compounds related to fruit ripening/spoilage/pathogenesis will indicate "key" time-points of physiological changes of the fruit. Those key points will be evaluated for cDNA library construction. Such libraries will be used to study differential gene expression by expression profiling using the DNA-micro array technology. The expression profiling will enable the analysis of thousands of genes, thus enabling the isolation of cDNA clones whose expression is related to spoilage/pathogenesis, thus maybe unravelling new metabolic pathways or differential regulations of described ones.
Other important insights about the production of natural compounds during plant-pathogen interaction have been achieved by P2 and P3 (University of Nijmegen ref. 3).
Together, our approaches provide a picture of fruit maturation and spoilage and Botrytis growth in vitro and in vivo on infected tomato fruits. Several storage parameters such as temperature and humidity, as well as the developmental stage of the harvested fruit, affect its quality and determine successful Botrytis infection and therefore fruit spoilage. So far, the traditional solutions to these problems have been stored under controlled or modified atmospheres with the use of synthetic pesticides. The understanding of the mechanism of fungal penetration and of the plant response to the pathogen attack will enable us to identify the components of the natural defence response in plants which can be used not only as early and sensitive indicators for spoilage, but which can also be optimised in order to enhance resistance.
main references:
1) Pashkoulov, et al. "Biochemical Characterisation of Polygalacturonases from five different isolates of Botrytis cinerea" Mycological Research, July 2002.
2) De Martinis and Benvenuto. "Botrytis cinerea Infection Activates Ethylene and Jasmonic Acid-Related Gene Expression in Harvested Tomato Fruits." Proceedings from the NATO: Advanced Research Workshop on Biology and Biotechnology of Plant Hormone Ethylene (in press).
3) Cristescu, et al. "Ethylene production by Botrytis cinerea in vitro and in tomato fruit" Applied and Environmental Microbiology, November 2002.
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