CN109917118B - Light-operated nondestructive detection method for fungus diseases of picked fruits and vegetables - Google Patents
Light-operated nondestructive detection method for fungus diseases of picked fruits and vegetables Download PDFInfo
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Abstract
The invention discloses a light-operated nondestructive detection method for fungal diseases of picked fruits and vegetables. The ethylene concentration detector can monitor the ethylene concentration in the sample sealing chamber connected with the ethylene concentration detector in real time. The sample sealing chamber is a transparent and light-transmitting sealed container, and a cassette is arranged outside the sample sealing chamber and is used when the sample needs dark treatment. A closed gas loop is formed between the ethylene concentration detector and the sample sealing chamber. The method takes the ethylene synthesis and light induction of fruits and vegetables with fungal diseases as the principle, and judges whether the fruits and vegetables are infected by the fungi or not by monitoring whether the sample to be detected has the phenomenon of rapid jump of the ethylene yield or not after the illumination condition is changed in real time under the illumination mode of 'first darkness and then white light'. The method can realize accurate early warning of fungal diseases of fruits and vegetables under the condition that the incidence of diseases is lower than 10% in the initial stage of diseases, realize timely treatment of infected fruits and vegetables to reduce the loss caused by fungal infection, and can be widely applied to early warning of fungal diseases of fruits and vegetables.
Description
Technical Field
The invention belongs to the technical field of fruit and vegetable disease diagnosis, and particularly relates to a light-operated detection device and a light-operated detection method for monitoring ethylene release amount of fruits and vegetables in real time, which are used for early warning of fungal diseases of picked fruits and vegetables, reducing disease propagation and spread, and reducing rot loss caused by the fungal diseases.
Background
Fruits and vegetables contain abundant water and various nutrient substances, and in the processes of storage, transportation and sale after picking, the quality of the fruits and vegetables is reduced and even the edible value of the fruits and vegetables is completely lost due to improper control of environmental conditions such as temperature, humidity and the like, pest and disease attack, pesticide residue and mechanical damage, so that huge economic loss is caused. The pathogenic fungi have the characteristics of long incubation period, strong infectivity, high resistance to bactericides and severe environments and the like, so that the fungal diseases become the most main factors causing economic loss after picking, and account for more than 70 percent of the total loss. Therefore, early detection, isolation and prevention of fungal diseases are the key to reduce the economic loss after harvesting, and the development of related technologies is very important.
The current commonly used fungal disease detection technology mostly uses tissue slices to judge whether a sample contains fungal tissues or not by culturing and observing the tissue slices under a microscope, or detects the cell membrane potential and diagnoses the fungal infection by using a PCR technology. However, the detection means all have the defects of complex instrument operation, higher technical requirement on operators, more expensive instruments, larger occupied area, high detection cost, long detection time and the like, so the application range is limited. More importantly, most of the existing fruit and vegetable disease detection methods are destructive detection of sampling, the accuracy of disease diagnosis of a whole batch of fruit and vegetable products may have deviation in detection results, and the detected samples cannot be sold continuously, so that the detection cost is increased. Therefore, a nondestructive detection means which is simple, convenient and feasible, low in cost and short in detection period still needs to be developed, and the method is widely used for early warning of postharvest fungal diseases.
Ethylene is the only gaseous plant hormone, produced in large quantities during late plant maturation, senescence abscission, and when subjected to mechanical damage and stress. Because the ethylene-removing agent can catalyze the softening, the maturing and the aging of fruits, the ethylene is usually removed after the fruits are picked, the aging of the fruits and the vegetables is further delayed, and the effect of keeping the fruits fresh is achieved. In addition, studies have shown that plants are subject to a substantial increase in ethylene release after being infected with fungi, and this portion of ethylene is called diseased ethylene. On the other hand, phytopathogenic fungi also synthesize ethylene and have three ethylene synthesis pathways. Among them, only Trichosporon mucosae (Dictyostelium mucoides) and Penicillium citrinum (Penicillium citrinum) have been reported to synthesize ethylene by the ACC (1-aminocyclopropane-1-carboxylate) pathway of the plant-like species; the EFE (ethylene forming enzyme) pathway is mostly found in non-phytopathogens; most phytopathogenic fungi synthesize ethylene via the KMBA (2-keto-4-methyl-thiopyric acid) pathway. The pathway uses methionine (Met) as a substrate and forms an intermediate product KMBA through transamination. The breakdown of KMBA is induced by light, forming ethylene in different ways under dark and light conditions: under light conditions, photolysis of KMBA is a rapid, enzyme-independent oxidation process that is regulated by redox-active substances such as Flavin Mononucleotide (FMN); under dark conditions, it is a slow oxidation process that relies on the action of the enzyme forming ethylene.
We prove that fungi participate in the synthesis of ethylene disease of a host fruit and vegetable-fungi interaction system, and the photoinduction rule that the synthesis rate of ethylene is higher under illumination than under darkness specially exists in the ethylene synthesis process of the disease interaction system, but the ethylene release of fruits and vegetables which are not infected by the fungi is not induced by illumination. Subsequently, we demonstrated that changes in the rate of ethylene synthesis in disease-interacting systems can be detected rapidly in the short term after changes in lighting conditions by dynamic detection of the ethylene synthesis process. Based on the principle, by dynamically monitoring the ethylene release amount change of a fruit and vegetable sample to be detected in the lighting mode of 'first darkness and then white light', the invention provides the method and the device for nondestructive detection of the postharvest fungal diseases, which have the advantages of quick detection, high sensitivity, simplicity, convenience, practicability and low cost, can accurately early warn the postharvest fungal diseases in the early development stage of the fungal diseases, and can provide technical support for preventing and controlling the spread of the postharvest fungal diseases of fruits and vegetables and reducing economic losses caused by fruit and vegetable deterioration.
Disclosure of Invention
The invention provides a light-operated nondestructive detection device for detecting the fungal diseases of picked fruits and vegetables, which can perform early warning on whether the fruits and vegetables have the fungal diseases or not under the condition of not damaging detected fruit and vegetable samples, evaluate the infection conditions of the detected fruits and vegetables, perform early warning at the early stage of disease development, effectively reduce the aggravation and spread of the fungal diseases, and is convenient to widely apply and popularize due to the advantages of simple operation, quick detection, low cost and the like.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention discloses a light-operated nondestructive detection device for fungus diseases of picked fruits and vegetables, which comprises: an ethylene concentration detector and a sample sealing chamber; the sample sealing chamber is internally provided with a sample sealing box, a light source and a light-shading cassette; the sample sealing box is communicated with the ethylene concentration detector; the light-resistant cassette can completely cover the sample sealing cartridge; the light source is an LED white light source.
In the invention, the ethylene concentration detector can continuously monitor the ethylene concentration in real time.
In the invention, the illumination intensity of the LED white light source is 20 mu mol.m -2 s -1 。
In the invention, the light source is fixed right above the top end outside the sample sealing box by using an insulating adhesive tape, the switch controls the opening and closing of the light source after the power supply is switched on, and the switch is positioned outside the light-resistant cassette after the sample sealing box is covered by the light-resistant cassette.
In the present invention, the sample capsule is transparent to all wavelengths of visible light.
In the invention, the sample sealing box is communicated with the ethylene concentration detector through a rubber hose, and the sample sealing box and the rubber hose are sealed through a rubber plug.
In the invention, under the illumination mode of 'first darkness and then white light', the rapid ethylene release amount sharp increase can be detected when the illumination condition is changed, so the light-shading cassette is characterized by being an opaque black plastic square box, the sample sealing box is arranged in the light-shading cassette and can completely cover the sample sealing chamber, the sample sealing box is in complete darkness under the state that the light source is closed, and the LED white light source is ensured to be the only light source which can be penetrated by the sample sealing box when the light source is opened.
The rubber hose is characterized in that the hose body and the interface are required to be good in air tightness, and the sample sealing chamber is connected with the air inlet and the air outlet of the ethylene concentration detector respectively and used for enabling the gas circulation to be formed between the sample sealing chamber and the ethylene concentration detector and ensuring smooth gas circulation to be realized between the sample sealing chamber and the ethylene concentration detector.
Based on the device for the light-operated nondestructive detection of the fungal diseases of the picked fruits and vegetables, the invention also provides a method for the light-operated nondestructive detection of the fungal diseases of the picked fruits and vegetables, which comprises the following steps:
the method comprises the following steps: selecting healthy and disease-free fruits and vegetables, placing the fruits and vegetables in a completely dark closed environment, monitoring the ethylene concentration in real time under the dark condition after the ethylene concentration of the closed environment is stable, and taking the detection result as the standard range of the ethylene release amount of healthy fruit and vegetable samples with the quality;
step two: randomly sampling and selecting the fruit and vegetable samples to be tested with the same quality as the step, and placing the samples in a completely dark closed environment;
step three: after the ethylene concentration in the closed environment is stable, monitoring the change of the ethylene concentration in real time under a dark condition;
step four: turning on a white light source, and monitoring the ethylene concentration change of the fruit and vegetable sample to be detected under the white light induction condition in real time;
step five: if the ethylene concentration of the fruit and vegetable sample to be detected maintains dynamic balance in a normal range, the fruit and vegetable sample to be detected is proved to have no disease; if the ethylene concentration is higher than the normal range but the dynamic balance is maintained, the fact that part of the fruit and vegetable samples to be detected have physical damage is proved; if the ethylene concentration sharply increases after the ethylene concentration is changed into white light from darkness, the fruit and vegetable sample to be detected is proved to have fungal diseases.
Preferably, in the first step, the monitoring time is 1 h; in the third step and the fourth step, the monitoring time is 30 min.
The invention has the following beneficial effects: according to the invention, the ethylene synthesis amount of the fruit and vegetable sample to be detected in the sealed chamber is dynamically monitored in the illumination mode of 'first darkness and then white light', whether the fruit and vegetable sample has rapid ethylene release amount increase after the illumination condition is changed is judged, and whether the fruit and vegetable sample has fungal diseases is further evaluated. By using the method, the fungal diseases can be quickly and accurately detected in the early development stage of the fungal diseases without damaging the sample, and the method is combined with related isolation sterilization measures, so that the problems of spread and large-area outbreak of the fungal diseases in the storage, transportation and sale periods of picked fruits and vegetables caused by untimely detection due to overlong detection time, high cost and the like can be effectively solved, and the postharvest loss of the fruits and vegetables caused by fungal infection can be further reduced.
Because the fungi can rapidly synthesize a large amount of ethylene under the illumination condition, and the invention requires that the rapid and sharp increase of the ethylene release amount of the fungi in the disease interaction system under the illumination mode of 'first darkness and then white light' can be sensitively detected, the detection of complete light shielding in the early stage and strong illumination stimulation in the later stage are very important for the accurate early warning of the fungal diseases. The invention also has the following advantages in meeting this requirement: firstly, the light-shielding cassette provided by the invention can enable the sample sealing chamber to be in an environment which can not be penetrated by external light, so that the complete light shielding under a dark condition and the uniqueness of the illumination source under an illumination condition are ensured, and further, the detection result is not interfered by the external light; secondly, the illumination intensity of the LED light source provided by the invention can ensure that fungi can rapidly decompose a large amount of KMBA to form ethylene, and the abnormal change of the temperature in the sample sealing chamber can not be caused in the detection process, so that the damage of the fruit and vegetable sample can be better ensured while the rapid identification of diseases is realized; thirdly, the ethylene concentration detector provided by the invention realizes real-time monitoring of the ethylene concentration, greatly simplifies the operation steps and the operation requirements compared with the traditional ethylene detection method of sampling at fixed time and measuring the concentration by using a gas chromatograph, more intuitively realizes visualization of the whole ethylene synthesis process of the light-adjusting disease interaction system, and improves the accuracy of the detection result.
Drawings
FIG. 1 is a schematic structural diagram of the device for optically-controlled nondestructive detection of fungal diseases of picked fruits and vegetables. (1, ethylene concentration detector; 2, sample sealing chamber; 3, sample sealing box; 4, 5, rubber plug; 6, external LED white light source; 7, light-proof box; 8, 9, rubber hose; arrow in the figure represents gas flow direction)
FIG. 2 shows the measurement of ethylene synthesis rate and ethylene release amount under dark and white light conditions for Colletotrichum gloeosporioides, Botrytis cinerea, Penicillium digitatum, Alternaria alternata and Fusarium asianum. Note: (A) ethylene synthesis rate of fungi under dark and white light conditions; (B) the (F) is the real-time detection result of the ethylene release amount of colletotrichum gloeosporioides, botrytis cinerea, penicillium, alternaria and fusarium asianum under the illumination mode of 'first darkness and then white light' (darkness (30min) -white light (30 min)).
FIG. 3 shows the growth of tomato systems and the real-time measurement of ethylene release in "first dark and then white light" lighting mode. Note: (A) - (D) are the real-time detection results of ethylene release amount under the illumination mode of 'first darkness and then white light' 1-3 days after treatment of a control group (healthy fruits without any treatment), a mechanical injury group (scratched by a sterilized scalpel), a freezing injury group (treated at the temperature of-20 ℃ for 12 hours) and an infection group (inoculated with a wound and botrytis cinerea tender fungus plate) in sequence; (E) the lesion area and disease index of the infection group 1 to 3 days after the inoculation treatment are counted; (F) is the growth status of each system 1-3 days after treatment.
FIG. 4 shows the growth of each lemon system and the real-time measurement of ethylene release in "first dark and then white light" lighting mode. Note: (A) - (D) are the real-time detection results of the ethylene release amount of the control group, the mechanical damage group, the freezing injury group and the infection group under the illumination mode of darkness (30min) -white light (30min) 1-3 days after treatment in sequence; (E) the scab area and the disease index of the infection group are counted 1 to 3 days after the inoculation treatment; (F) is the growth condition of each system after 1-3 days of treatment.
FIG. 5 is a sensitivity test for testing grape fruit gray mold simulating natural onset. Note: each row in the figure is sequentially the fruit morbidity, the fluorescent microscope observation (the ruler is 20 mu m) of the hypha growth condition at the fruit stalk border pulp and the real-time detection result of the ethylene release amount under the illumination mode of 'first darkness and then white light' from left to right
FIG. 6 is a schematic structural diagram of the method for light-operated nondestructive detection of fungal diseases of picked fruits and vegetables.
Detailed Description
The invention is described in further detail in connection with the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
The plant pathogenic fungi mainly synthesize ethylene by a KMBA way, Met is used as a substrate in the way, an intermediate product KMBA is formed under the action of transamination, and then the KMBA is decomposed into ethylene, carbon dioxide and volatile sulfide. The KMBA has different decomposition mechanisms under the light and the dark, the KMBA is slowly oxidized and decomposed to form ethylene under the action of enzyme under the dark, and the decomposition of the KMBA under the light condition is a rapid photolysis process without enzyme, so that the ethylene synthesis of fungi is induced by light, and the synthesis rate is higher under the light condition. Plants release a large amount of diseased ethylene after suffering from fungal infection and disease, and the examples demonstrate that the release of diseased ethylene is also light induced, which is not present on fruits and vegetables not infected by fungi. According to the method, whether the ethylene release amount of the fruits and vegetables to be detected is rapidly increased after the illumination condition is changed from darkness to white light is dynamically monitored, whether the fruits and vegetables to be detected are infected by fungi is further warned, the occurrence of the fungal diseases is simply, rapidly and inexpensively detected under the condition that the samples are not damaged, and the aggravation and the spread of the fungal diseases caused by the defects that the detection is not timely, the sample capacity is not large enough, the sensitivity of the detection method is not high and the like are reduced.
The light-operated nondestructive detection device for detecting the fungal diseases of the picked fruits and vegetables in the embodiment comprises an ethylene concentration detector 1, a sample sealing chamber 2 and rubber hoses 8 and 9 which connect the ethylene concentration detector and the sample sealing chamber. Wherein, sample seal chamber 2 includes sample seal box 3, rubber buffer 4 and 5, external LED white light source 6, light-resistant magazine 7.
In this example, the ethylene concentration detector 1 can continuously detect the ethylene concentration in the sample-sealed chamber in real time.
In this example, the sample sealing box 3 is only communicated with the ethylene concentration detector, is completely sealed from the external environment, and can transmit all the wave bands of visible light.
In this example, rubber buffer 4 and rubber buffer 5 are for being used for establishing being connected of sample seal box 3 and rubber hose 8 and rubber hose 9 to guarantee that the gas tightness of sample seal box is good.
In this example, the white light can rapidly induce the ethylene synthesis of the contaminated fruits and vegetables in large quantities, so the external LED white light source 6 has an illumination intensity of 20 μmol m -2 s -1 The white light stimulation device is used for giving white light stimulation to fruits and vegetables in the sample chamber.
In the embodiment, the method for detecting the fungal diseases of the picked fruits and vegetables by using the light-operated nondestructive test comprises the following steps:
the method comprises the following steps: determining the ethylene release amount of healthy fruits and vegetables: selecting a proper amount of healthy and disease-free fruits and vegetables, placing the fruits and vegetables in a sample sealing box, covering a light-shielding cassette outside the sample sealing box to enable the samples to be in a completely dark state, sealing for 1h until the ethylene concentration in the box is stable (the sealing time can be properly increased or shortened due to the types and the quantity of the samples), opening an ethylene concentration detector, monitoring the ethylene concentration in the sample sealing box in real time under the dark condition, wherein the detection time is 1h, and the detection result is used as the standard range of the ethylene release of the fruit and vegetable samples with the quality (the step is repeated for at least 3 times at different sampling times due to different maturity degrees of the fruits and vegetables);
step two: randomly sampling and selecting the fruit and vegetable samples which are equal to the fruit and vegetable samples in the first step, placing the fruit and vegetable samples in a sample sealing box, covering a light-resistant cassette outside the sample sealing box to enable the samples to be in a completely dark state, and sealing for 1 hour till the ethylene concentration in the box is stable, wherein the sealing time can be properly increased or shortened due to the types and the quantity of the samples;
step three: after the ethylene concentration in the sample sealing box is stable, starting an ethylene concentration detector, and monitoring the change of the ethylene concentration in the sample sealing chamber under the dark condition in real time, wherein the detection time is 30 min;
step four: after real-time monitoring is carried out for 30min under a dark condition, an LED white light source is turned on, ethylene concentration change in a sample sealed chamber under a white light induction condition is continuously monitored in real time, and the detection time is 30 min;
step five: and drawing a trend graph by taking the detection time (min) as an abscissa and the ethylene concentration (ppm) in the sample sealing box as an ordinate, wherein the standard for judging whether the sample is infected by the fungi is as follows: if the ethylene concentration maintains dynamic balance in a normal range, the sample is proved to have no disease; if the ethylene concentration is higher than the normal range but the dynamic balance is maintained, part of the sample is proved to have physical damage, such as mechanical damage or extreme temperature stress; if the ethylene concentration sharply increases after the dark is converted into white light under the illumination condition, the sample is proved to have fungal diseases.
The invention dynamically detects the ethylene release amount of five typical plant pathogenic fungi under the illumination mode of 'first darkness and then white light', proves that the rule of rapid and sharp increase of light-induced ethylene synthesis amount has universality in the plant pathogenic fungi, then proves that the rule of rapid and sharp increase of light-induced ethylene synthesis amount only exists specifically in fungi-infected fruits and vegetables by taking a physical damage system and a fungal disease system of tomatoes and lemons as research objects, simulates the process of grey mold explosion of grapes in a natural state in a non-wound inoculation mode, and verifies the feasibility, sensitivity and accuracy of the invention.
Example I white light induces the rapid and large-scale synthesis of ethylene by phytopathogenic fungi
Five typical plant pathogenic fungi are selected in the example, wherein colletotrichum gloeosporioides is the main pathogenic fungi of tropical fruits and citrus fruits; botrytis cinerea is an important necrotic pathogen, can infect more than 2000 plants, and is the second largest pathogenic fungus in the world after rice blast; the penicillium can cause penicilliosis of various fruits such as oranges, grapes, apples and apricots, and conidia generated by the penicillium is also an allergen, so that anaphylactic reaction of partial crowds is caused; fusarium Asiatica can infect cereal crops and vegetables such as asparagus, eggplant and the like, and the pathogenic factor DON toxin generated by the Fusarium Asiatica is the second largest toxin after aflatoxin, which can cause symptoms such as vomit, dizziness, fever and the like and even threaten life. Therefore, the detection and the prevention of the five pathogenic fungi after the picking are particularly important. In the selected strains in the example, the botrytis cinerea is a model strain B05.10; the colletotrichum gloeosporioides EX-16-01 strain is obtained by separating from the surface of lime; the penicillium Pen-1 strain is separated from the surface of the navel orange; alternaria alternata EX-16-07 strain is separated from the surface of tomato; the Fusarium Asiatica Exap-08 strain is separated from the surface of Asparagus officinalis.
For the detection of the average rate of ethylene synthesis, 1mL of 10 concentration was used 7 Mixing the spores in 99mL of 55 deg.C PDA +10mM Met culture medium, mixing, pouring, culturing at 23 deg.C for 48 hr, punching 10 bacteria dishes with 5mM diameter puncher, placing in 10mL penicillin bottle, plugging the bottle with rubber stopper, culturing under LED white light or dark condition for 4 hr, extracting 1mL gas with syringe, and culturing with GC9800 typeThe FID detector of the gas chromatograph measures the ethylene concentration and calculates the average rate of bacteroid disk ethylene synthesis in μ L/h in the bottle. The results are shown in FIG. 2 (A): the ethylene synthesis rate of each strain under white light is higher than that of the detection value under dark condition, which indicates that the test strains synthesize ethylene in KMBA way, and the synthesis is induced by light.
We then monitored the light-induced ethylene synthesis dynamically using the "continuous mode" mode of a FELIX F-950 Three Gas Analyzer hand-held ethylene detector. Specifically, 100. mu.L of 10-concentration PDA + Met medium with a diameter of 90mm and laid with cellophane is dripped 6 The spore suspension/mL is evenly coated by a sterilized coating rod, and is cultured for 48 hours at 23 ℃ under white light after being dried. The petri dish was then sealed in the dark for 1h in a sample-sealed chamber with a volume of 2500mL, and then the sample-sealed chamber was illuminated in an illumination mode of "dark first and white light" (dark (30min) -white light (30min)) and the ethylene concentration in the sample chamber was monitored in real time, and treated in full darkness as a control. And scraping hyphae after detection, weighing the dry weight of the hyphae, and finally calculating the dynamic change of the hyphae ethylene synthesis amount in unit mass, wherein the unit is mu L/g. The results are shown in FIGS. 2(B) to (F): the ethylene synthesis amount of the control group slowly increased and then maintained a stable dynamic equilibrium at a lower level, but when the light condition was changed from dark to white, the ethylene synthesis amount rapidly increased, and reached a peak within 10min and then a new dynamic equilibrium state was formed at a higher level. Therefore, we achieved visualization of the entire process of light-induced fungal ethylene synthesis and demonstrated that white light induces fungi to synthesize ethylene rapidly and in large quantities.
Example two, dynamic monitoring of ethylene synthesis in tomato systems under "dark-first then white" light mode.
Soaking fructus Lycopersici Esculenti in 75% ethanol for 30s before processing, cleaning with sterile water twice, and air drying on clean bench. In this example, 4 experimental groups were set up, respectively: control, healthy fruit without any treatment; performing scratch treatment on the surface of a fruit by using a sterile scalpel in a mechanical injury group; freezing damage group, and treating the fruits at-20 deg.C for 12 hr; and (4) infecting groups, namely, wounding and inoculating tender botrytis cinerea bacterium dishes. After treatment, each experimental group was cultured in the dark at 23 ℃. And then, placing each experimental group in a sample sealing chamber with the volume of 2500mL for 1h every 24h in the dark, then irradiating the sample sealing chamber in an illumination mode of 'first darkness and then white light' and monitoring the ethylene concentration in the sample chamber in real time. Each experimental group was weighed and the results were converted into changes in the amount of ethylene synthesized per unit mass of the fruit, in μ L/kg.
The growth status after treatment and the disease progression of the affected groups in each experimental group are shown in FIGS. 3(E) and (F). The ethylene synthesis amount of the control group was maintained at a level of about 18. mu.L/kg in all 3 days of the test, as shown in FIG. 3(A), and was not induced by light. Ethylene release from the mechanically damaged and frozen groups as shown in fig. 3(B) and (C), even though physical damage increased the ethylene release from tomato fruits, ethylene release was not light induced. The detection result of the "Botrytis cinerea-tomato" interaction system is shown in FIG. 3(D), and the disease spots are already visible at the inoculation site on the first day after treatment, and the rapid ethylene release burst after the illumination condition is changed into white light can be detected, which is the same as the monitoring result rule of the Botrytis cinerea in-vitro experiment. And the phenomenon of photoinduced ethylene release becomes more and more obvious along with the development of the disease. Therefore, the phenomenon that white light induces the disease ethylene to be rapidly released exists specifically in the fungal disease system of the tomato.
EXAMPLE III, dynamic monitoring of ethylene Synthesis in lemon systems in the "dark-first then white" light mode
The treatment method of the lemon fruits and the treatment method of each experimental group are the same as the second example, and the infection group is inoculated with the young fungus plate of colletotrichum gloeosporioides. As shown in fig. 4(a), healthy lemon fruits release only trace amounts of ethylene. The ethylene release rule of each system of the lemon fruit is the same as that of each system of the tomato, namely, the ethylene synthesis of the fruit without fungal infection is not induced by short-term illumination, and the ethylene of the fruit with bacteria is rapidly and massively synthesized after the fruit with bacteria is stimulated by white light. Combining the results of examples two and three, it can be confirmed that the ethylene outbreak caused by fungal infection is induced by light, and the rule exists only in fruits and vegetables with fungal diseases, and can be used as a basis for diagnosing postharvest fungal diseases.
Example four, "Botrytis cinerea-grape" interaction system to simulate natural onset of disease ethylene synthesis dynamic monitoring in "dark-first then white light" illumination mode
In a natural state, conidia of botrytis cinerea usually invade plants through natural orifices of the plants or wounds caused by mechanical damage, and further germinate to form mycelia, and infect plant cells to cause large-area cell death. The experiment simulates the natural infection condition of botrytis cinerea, after the test grapes are disinfected, cleaned and air-dried, the stems of the grapes are placed upwards in a culture box, and 10 mu L of the test grapes with the concentration of 10 mu L are dripped into the stems of the grapes 6 A/mL suspension of GeP-labeled Botrytis cinerea spores, followed by dark cultivation at 23 ℃. The following daily ethylene monitoring and release amount calculation methods were the same as in example two. After the treatment, the tissue around the fruit stem was peeled every 24 hours, and the growth of hyphae in the tissue was observed under a fluorescent microscope. As can be seen from the results of the photograph and the microscopic observation, no lesion was visible on the first day of the inoculation treatment, but the spores had invaded the grape tissue and had germinated to form a mycelium; the incidence rate on the following day was 48.6%, but the lesion diameter was less than 4mm, and the whole disease was still in the initial stage of the disease, i.e., the lesion was small and did not constitute a cross infection. The detection result of the ethylene release amount shows that the ethylene outbreak of the disease induced by white light can be detected in the next day after the treatment, and the phenomenon is more and more obvious along with the outbreak of the disease. Therefore, the method can be used for accurately diagnosing the occurrence of the fungal diseases when only a few samples are attacked in grape samples and diseases are initially developed.
In conclusion, the invention develops a method and a device for dynamically detecting the ethylene release amount of a sample to be detected; the ethylene synthesis of the in vitro cultured fungi and the fungi-infected fruits is rapidly induced by illumination through the ethylene release condition of the in vitro cultured colletotrichum gloeosporioides, botrytis cinerea, penicillium, alternaria alternata and fusarium asianum, and tomato and lemon fruits, a physical damage system and a fungus disease system under the illumination mode of 'first darkness and then white light', and the rule does not exist in fruits which are not infected by the fungi, so the ethylene synthesis can be used as a basis for diagnosing the fungus diseases of fruits and vegetables after picking. Subsequently, in order to prove the accuracy, the sensitivity and the feasibility of the method, the process of infecting plant tissues under the natural condition of botrytis cinerea is simulated in a non-invasive grape fruit stalk inoculation mode, the morbidity process of grapes is recorded by means of photographing and microscopic observation, and whether the ethylene release of the grapes is induced by light or not is monitored. The result shows that the invention can accurately diagnose the occurrence of fungal diseases at the early stage of the disease with low incidence and unobvious diseases. The specific diagnosis method comprises the following steps: selecting a fruit and vegetable sample to be detected through random sampling, placing the fruit and vegetable sample in a sample sealing box, sealing the sample sealing box in the dark for 1h, then giving illumination stimulation of 'first darkness and then white light' to the sealing box, detecting the ethylene release amount in the sealing box in real time, if the ethylene concentration in the box is rapidly and greatly increased after the illumination condition is changed from the dark to the white light, fruits which are infected by fungi and have been diseased exist in the detected sample, and treating the fruit and vegetable by matching with related isolation and sterilization measures so as to reduce the risk of fungal disease outbreak. The sealing and detecting time can be shortened or prolonged properly according to different detecting objects.
The protection content of the present invention is not limited to the above examples. Variations and advantages that may occur to those skilled in the art are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be protected by the following claims.
Claims (9)
1. A light-operated nondestructive detection method for fungus diseases of picked fruits and vegetables is characterized by comprising the following steps:
the method comprises the following steps: selecting healthy and disease-free fruits and vegetables, placing the fruits and vegetables in a completely dark closed environment, detecting the ethylene concentration in real time under the dark condition after the ethylene concentration of the closed environment is stable, and taking the detection result as the standard range of the ethylene release amount of healthy fruit and vegetable samples with the quality;
step two: randomly sampling and selecting the fruit and vegetable samples to be tested with the same quality as the step, and placing the samples in a completely dark closed environment;
step three: after the ethylene concentration in the closed environment is stable, monitoring the change of the ethylene concentration in real time under a dark condition;
step four: turning on a white light source, and monitoring the ethylene concentration change of the fruit and vegetable sample to be detected under the white light induction condition in real time;
step five: if the ethylene concentration of the fruit and vegetable sample to be detected maintains dynamic balance in a normal range, the fruit and vegetable sample to be detected is proved to have no disease; if the ethylene concentration is higher than the normal range but the dynamic balance is maintained, the fact that part of the fruit and vegetable samples to be detected have physical damage is proved; if the ethylene concentration sharply increases after the ethylene concentration is changed into white light from darkness, the fruit and vegetable sample to be detected is proved to have fungal diseases.
2. The method for light-operated nondestructive testing of the fungal diseases of the harvested fruits and vegetables as claimed in claim 1, wherein the method employs a light-operated nondestructive testing device for the fungal diseases of the harvested fruits and vegetables, the device comprising: an ethylene concentration detector and a sample sealing chamber; wherein a sample sealing box, a light source and a light-shading cassette are arranged in the sample sealing chamber; the sample sealing box is communicated with the ethylene concentration detector; the light-shielding cassette can completely cover the sample sealing box, so that the sample sealing box is in an absolutely light-tight environment; the light source is an LED white light source.
3. The light-operated nondestructive detection method for the fungal diseases of the picked fruits and vegetables as claimed in claim 2, wherein the ethylene concentration detector can continuously monitor the ethylene concentration in real time.
4. The light-operated nondestructive detection method for fungal diseases of picked fruits and vegetables as claimed in claim 2, wherein the illumination intensity of the LED white light source is 20 μmol-m -2 s -1 。
5. The light-operated nondestructive detection method for the fungal diseases of the picked fruits and vegetables as claimed in claim 2, characterized in that the light source is fixed right above the outside of the sample sealing box by an insulating adhesive tape, and the opening and closing of the light source is controlled by a switch; after the sample sealing box is covered by the light-shielding cassette, the switch is positioned outside the light-shielding cassette.
6. The light-operated nondestructive testing method for the fungal diseases of the picked fruits and vegetables as set forth in claim 2, characterized in that the sample sealing box can be penetrated by all wave bands of visible light.
7. The light-operated nondestructive testing method for the fungal diseases of the harvested fruits and vegetables as claimed in claim 2, characterized in that the sample sealing box is communicated with the ethylene concentration detector through a rubber hose to form a closed gas circulation.
8. The light-operated nondestructive testing method for the fungal diseases of the picked fruits and vegetables as claimed in claim 7, wherein the sample sealing box and the rubber hose are sealed by a rubber plug.
9. The light-operated nondestructive detection method for the fungal diseases of the picked fruits and vegetables as claimed in claim 1, wherein in the first step, the monitoring time is 1 h; in the third step and the fourth step, the monitoring time is 30 min.
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| CA2352639A1 (en) * | 2000-07-14 | 2002-01-14 | John Joseph Cullen | A method and apparatus for monitoring a condition in chlorophyll containing matter |
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