WO1996038582A1

WO1996038582A1 - A process for the early detection of infection in a crop

Info

Publication number: WO1996038582A1
Application number: PCT/IE1996/000035
Authority: WO
Inventors: Peter Wyn Jones
Original assignee: University College Cork
Priority date: 1995-06-01
Filing date: 1996-05-31
Publication date: 1996-12-05
Also published as: AU5909296A

Abstract

The invention provides a method for the early detection of infection within a growing crop by contacting crop organ samples such as leaves with a senescence material and incubating the crop organ samples under controlled conditions to accelerate infection. This allows a farmer to take curative steps more quickly than if the natural development of the infection was awaited. The invention also provides an enclosure provided by an incubator (1) containing sample holder mounts (13), a heater (11), a light source (4, 5) and a humidity controlling membrane (23) for the sample holders. The base (10) of the incubator (1) forms a reservoir for senescence retardant liquid.

Description

"A Process for the Early Detection of Infection in a Crop"

This invention relates to a process for the early detection of infection within a crop. It further relates to an apparatus used in this process.

Crop infection is a major problem for farmers at certain times of the year. If it goes untreated, infection can result in serious yield losses, if not the complete destruction of the crop.

There are three principal strategies currently adopted regarding the treatment of crops by fungicide spray applications .

The first strategy involves spraying the crop several times per year when the crop reaches specific stages of development. These stages generally represent the beginning of the most vulnerable phases of the plant life cycle to yield less due to infection. The advantage of this strategy is that the crop is protected during the most important phases of development. However, there are several disadvantages. The strategy does not allow for seasonal differences in disease development. It may result in yield losses if an epidemic starts earlier than expected or result in unnecessary applications of fungicide with consequent unnecessary costs in fungicide, labour and threat to the environment, if the pathogen is not present or present only at low levels at the time of spraying. This prophylactic strategy also requires the use of broad-spectrum fungicides because the farmer very often does not know which diseases are present. 2 -

The second strategy involves the spraying of the crop when the disease reaches a specific threshold infection level, for example when 5% of the area of the uppermost two leaves of barley crop are affected by powdery mildew. This strategy has the advantage that sprays are only applied when needed, and a fungicide appropriate to the particular pathogen can be selected. There are many disadvantages. By the time the symptoms appear, crop losses are accruing. In addition, any delay such as caused by unsuitable weather conditions for spraying which is common during the critical spring/early summer period, will result in severe crop losses.

The third strategy involves spraying when weather conditions associated with the occurrence of infection persist for a critical period of time. This has the advantage of facilitating fungicide applications when necessary. However, very few diseases can be predicted in this way by weather conditions.

Several companies have developed immunological diagnostic kits which detect crop infection, or permit distinction between closely related diseases. These ELISA (Enzyme Linked Immunosorbent Assay) kits are highly sensitive and can detect crop infection very early on. However, they have a number of disadvantages. They are expensive and are not designed to handle large sample sizes. Often, because they are so sensitive, the results they give can be difficult to interpret. Quantification does not permit distinction to be made between a large number of infection foci at an early stage of development, and a small number of well-developed infection foci. In addition, as they work by means of a specific antibody-antigen reaction, they tend to be disease specific. Thus, in order to adequately test a crop for disease, a number of different disease specific ELISA kits are required. The invention seeks to provide a method and apparatus for the early detection of infection within crops which is suitable for large sample numbers, economical, practical,quantitative and capable of detecting any fungal pathogen present within the sample.

In accordance with the invention, this object is accomplished by a method for the early detection of infection a crop comprising the steps of:

taking a sample from the crop;

accelerating the development of infection in the crop sample under control conditions; and

detecting the presence of accelerated infection in the crop sample.

The method carried out in accordance with the invention has a number of advantages . By accelerating the development of infection in the samples, the development of any plant pathogens which may be present in the samples is accelerated. This allows for both the early detection of infection in the samples and the consequent early initiation of crop protection as appropriate. The method of the invention allows for the accelerated development of all plant pathogens . It is also practical in use and very economical.

The invention provides prior notice of an impending disease attack. It provides direct information on the presence and level of disease in the crop. It also provides a broad time window in which the crop can be sprayed so that any delay in spraying due to adverse weather conditions is less critical. In addition, the symptoms which develop are identical to those in the field, and allow for unequivocal identification of the disease(s) present.

In an especially preferred embodiment of the invention, the control conditions include the step of retarding the senescence of the crop sample, preferably by contacting the crop sample with a senescence retardant material. This is extremely important as it delays the ageing of the crop sample, especially plant organs such as leaves. The time for which the crop samples can be incubated is thereby extended.

In this specification, the term sample particularly refers to organs which have been detached from plants of a growing crop. The organs are preferably leaves, however they may also be pods, spikes, stem bases etc. Generally, when a sample is taken from a crop, a number of plants in the crop will be chosen and a number of organs will be detached from each plant.

Ideally, the development of infection in the crop sample is accelerated by incubating the crop sample under control conditions.

In one embodiment of the invention, the development of infection in the crop sample is accelerated by illuminating the crop sample under control conditions, preferably for between 12 and 24 hours per 24 hour incubation period.

Typically, the control conditions include temperature and humidity controls.

Preferably, the senescence retardant material is in a liquid form and may be applied to the crop sample by a contacting capillary material, preferably in a continuous manner. This is a simple and effective arrangement for automatically applying the senescence retardant liquid to the samples during incubation.

Preferably, the senescence retardant material is of the type which retards the growth of saprophytic fungi. As saprophytic fungi are not plant pathogens, their presence during the process is undesirable.

Preferably, the senescence retardant material is benzimidazole. This is a particularly good type of senescence retardant liquid.

The samples are maintained at between 10°C and 40°C. This temperature range allows the accelerated growth of both cool weather and warm weather pathogens.

Preferably, the samples are maintained at between 16°C and 25°C. This temperature allows for the improved accelerated growth of both cool weather and warm weather pathogens.

The samples may in some cases be maintained at a substantially constant temperature of for example 25°C. This simplifies the operation of the equipment while facilitating the growth of more serious warm weather pathogens.

Some of the samples may be maintained in the high humidity range and/or some are maintained in the low humidity range. This allows for the accelerated growth of high relative humidity pathogens like Septoria nodorum and/or low relative humidity pathogens like Erysiphe σraminis f.sp tritici. In one embodiment of the invention the crop samples are divided into at least two cohorts and the development of infection in one cohort of crop samples is accelerated in different control conditions than the control conditions for accelerating the development of infection in another cohort.

In this case typically the development of infection in one cohort of crop samples in accelerated at relatively low humidity conditions and the development of infection in another cohort of crop samples is accelerated at relatively high humidity conditions.

For ease of use the development of infection in the crop sample is accelerated in an incubator.

Preferably, the level of infection is assessed by:

identifying the disease present;

calculating the number of samples infected;

calculating the area of each sample which is infected; and

recording the period before which infection symptoms appeared.

These steps allow the user to accurately ascertain whether the crop from which the sample originated needs treating or not.

Preferably the method also includes the steps of:-

comparing the level of accelerated infection in the crop sample against predetermined standards; and assessing crop protection requirements accordingly.

The invention also provides a method for protecting a crop against infection comprising the steps of:-

early detection of infection in the crop by a method as claimed in any preceding claim; and

applying protection to the crop based on the early detection of the infection.

Preferably, the protection comprises the application of one or more fungicides.

The invention further provides a method for testing a crop including the step of early detection of infection in the crop by a method of the invention.

The invention also provides test apparatus for carrying out the method of the invention comprising an enclosure having:-

a crop sample holder;

a sample holder mount; and

control means for accelerating the development of infection in a crop sample mounted in the crop sample holder.

This is a practical, effective and easy to use apparatus for carrying out the process of the invention.

Preferably the enclosure is an incubator having a heater and preferably also a light source. This is a relatively simple construction of equipment for carrying out the method of the invention.

This is a practical, effective and easy to use apparatus for carrying out the method of the invention.

Preferably, there is provided a senescence retardant material holding and distribution means. This allows the incubation to be carried out completely automatically.

Preferably, the senescence retardant material holding and distribution means comprises a tray having a reservoir for senescence retardant liquid located below the sample holder mount whereby on mounting, the sample holder projects into the reservoir. This allows for ease of contact between the reservoir of senescence retardant liquid and samples in the sample holders and thus facilitates application of the senescence retardant liquid by capillary action.

Preferably the sample holder is constructed and arranged to receive a crop sample and a capillary material. Typically the sample holder comprises a back plate and a front plate which are spaced apart to receive the sample and a capillary material therebetween and clip means for the plates. This arrangement ensures that the samples are in continual contact with the capillary material during incubation.

Preferably the front plate has a recessed portion incorporating a plurality of inwardly projecting sample support dimples. This arrangement keeps the samples supported while allowing air to circulate around the samples. The recessed portion may incorporate a plurality of through holes which are covered by a humidity control means which may be provided by a humidity controlling membrane. This allows the humidity of the air around the samples to be controlled. In some cases for simplicity, the humidity membrane may not be fitted.

The invention will be more clearly understood from the following description of a process and an apparatus for carrying out the process, according to the invention, given by way of example only with reference to the accompanying drawings in which:-

Fig. 1 is a perspective view of an incubator used in carrying out the process according to the invention;

Fig. 2 is an exploded perspective view of the incubator;

Fig. 3 is a front perspective view of a sample holder;

Fig. 4 is a rear perspective view of the sample holder of Fig. 3;

Fig. 5 is a front perspective view of a sample holder with a portion cut away;

Figs. 6 to 10 are graphs illustrating the results of Field Trial No. 5.

The first step of the process involves the collection of samples from a field crop. The samples are usually leaves which are detached at a specific development stage. This step is carried out at intervals during risk periods, the intervals ranging from 7 days in early Summer to 14 days in Spring. Once a representative sample has been collected, it is incubated under controlled temperature and humidity conditions. , For detection of cool weather pathogens, the temperature will be kept at approximately 16°C whereas the temperature of approximately 25°C is preferable for detection of warm weather pathogens. During incubation, a senescence retardant liquid, in this case benzimidazole, is applied to the samples by a contacting capillary material. If the samples are infected, visible and easily identifiable symptoms will be produced within 7 days. This is up to 39 days ahead of symptoms developing on the corresponding field-grown plants. The infected samples will be accessed to ascertain the disease present, the percentage sample which develop symptoms, the average percentage symptom area per sample and the incubation period before symptoms appeared. These data can be used to monitor the progress of infection within a crop. When the extent of infection within the crop reaches threshold levels, the farmer will then usually have at least an extra two weeks in which preventative measures can be taken. For some diseases, e.g potato blight,the extra time may be less than two weeks. This advance treatment allows for better control of the infection.

It is understood that while the process of the present invention is ideal for foliar diseases of wheat and barley, it has also proved effective with cereal heads, oil seed rape pods, and the leaves of potato, oil seed rape, and Jerusalem artichoke.

An incubator used in carrying out the process according to the invention will now be described with reference to Figs. 1 to 5. The incubator 1 comprises an open mouthed rectangular box 2 and a closing lid 3 within which is mounted one or a number of fluorescent light bulbs 4 above a light diffuser 5. The open mouthed rectangular box 2 has opposing side walls 6, opposing end walls 7, one of which incorporates a draining hole 8 and a draining hole plug 9 and a base 10 which supports a thermostatically controlled heating element 11. The base 10 forms the bottom of a reservoir for a senescence retardant ιliquid into which is placed a sample holder support tray 12. The end walls 7 of the open mouthed rectangular box 2 incorporates a number of sample holder mounts 13 formed from guide rails 14 between which are mounted sample holders 15. The reservoir is designed so that the heated senescence retardant liquid can move freely between the six sample holder support trays.

Referring to Figs . 4 and 5, the sample holder 15 comprises two contacting plates, a back plate 16 which is attached to a front plate 17 by a hinge 18 situated at the top of each plate. The back plate 16 has a raised portion 19 which engages with a corresponding recess 20 on the front plate 17. The recess 20 has a plurality of inwardly projecting sample support dimples 21 and a number of through holes 22. A humidity-controlling low moisture vapour transport membrane 23 is provided to cover the through holes 22. When the membrane 23 is in place, a high relative humidity environment is created in the sample holder 15, otherwise a low relative humidity environment is created. In Fig. 5, a number of samples, in this case, leaves 24, are shown mounted on a sheet of capillary paper 25 sandwiched between the back plate 16 and the front plate 17.

In use and referring specifically to Fig. 5, the sheet of capillary material 25 is soaked in senescence retardant material and placed on the raised portion 19 of the back plate 16. The leaves 24 are placed on the capillary material 25 and the front plate 17 is placed over the leaves clipping on to the back plate 16. The leaves 24 are thus held against the capillary material 25 by the dimples 21 on the front plate. The humidity controlling membrane 23 may or may not be applied to the through holes 22. This depends on the relative humidity required in the sample holder 15.

Each of the sample holders 15 is then mounted in the incubator 1 on the guide rails 14 and the sample holder support tray 12 with the sample holder base 27 projecting towards the base 10 and hence into the reservoir which is filled with senescence retardant liquid. The temperature of the liquid is controlled by the thermostatically controlled heating element 11.

It will be appreciated that the relative humidity within the sample holders 15 may be varied by applying different combinations of the humidity controlling membrane 23 and/or by applying or omitting the membrane.

Various field trials were carried out on the apparatus according to the invention, details of which are given below. In the following trials the maximum daily temperatures varied from 15°C to 31°C and the minimum daily temperature varied from 5°C to 17°C.

Field Trial No. 1

CROP: Winter Wheat cv. Guardian

DISEASE: Powdery Mildew Ervsiphe αraminis f.sp tritici

TISSUE: Top two leaves Week

Detection Disease 4 5 6 7 8 9 System Parameter

% leaves infected 0 0 2 11 29 58

Field

Pustule no. per 0 0 0.05 0.55 2.15 4.71 leaf

% leaves infected 3 8 17 41 60 79

Invention Pustule no. per 0.08 0.41 1.11 3.72 7.12 8.62 leaf

Days to symptoms 6 3 0 0 0 0

The test was carried out and demonstrates quite clearly that the incidence of Erysiphe σraminis f.sp tritici

(powdery mildew) was at a serious level, namely 17% when a visual inspection in the field showed relatively little or no infection, and indeed as will be seen in the previous week, no infection showed in the field, but there was already a reasonably serious level of infection. It generally has been agreed that when the mildew infection gets to about 10% of the leaves being infected, it is essential to spray and obviously ideally the crop should be sprayed once the threshold level of infection is detected.

Field Trial No. 2

CROP: Winter Wheat cv. Guardian DISEASE: Septoria tritici TISSUE: Top two leaves Week

Detection Disease 1 2 3 4 5 6 7 System Parameter

% leaves infected 0 0 0 0 5 11 19

Field

Lesion no. per leaf 0 0 0 0 0.07 0.29 0.68

% leaves infected 10 8 15 21 31 41 53

Invention Lesion no. per leaf 0.16 0.13 0.60 1.03 1.14 2.31 3.19

Days to symptoms 6 6 4 1 0 0 0

What is interesting about these tests is that even when there was infection that it was not detected by field trials in any of the first four weeks of these tests. What is also interesting is that visual inspection in the field, for example, in week 7 showed a relatively small amount of lesions per leaf in those infected while the apparatus according to the present invention showed that over half the plants were about to suffer serious difficulties.

Field Trial No. 3

CROP: Spring Barley cv. Triumph

DISEASE: Rhvnchosporium secalis

TISSUE: Top two leaves Week

Detection Disease 1 2 3 4 5 6 7 System Parameter

% leaves infected 0 0 0 0 4 11 13

Field

Lesion no. per leaf 0 0 0 0 0.09 0.26 0.35

% leaves infected 4 5 12 16 20 25 22

Invention Lesion no. per leaf 0.05 0.09 0.21 0.42 0.81 1.11 1.16

Days to symptoms 5 4 2 1 0 0 0

The results of the trials on spring barley for Rhvnchosporium secalis are very interesting in that any farmer depending on visual inspection in the field would almost certainly have been exceptionally happy for much of the period of this trial when there was indeed a relatively large percentage of the crop being infected.

Field Trial No. 4

CROP: Spring Barley cv. Triumph

DISEASE: Powdery mildew ["Erysiphe graminis f.sp hordei)

TISSUE: Top two leaves

Week

Detection Disease 2 3 4 5 6 7 8 System Parameter

% leaves infected 0 0 5 19 23 35 47

Field

Pustule no. per 0 0 0.13 0.46 0.98 1.57 3.29 leaf

% leaves infected 6 13 29 48 73 87 94

Invention Pustule no. per 0.12 0.29 0.73 1.14 2.91 4.08 5.11 leaf

Days to symptoms 4 1 0 0 0 0 0 In detecting Erysiphe graminis f.sp hordei (powdery mildew) in spring barley what is interesting is that in week 4 when there was apparently only 5% incidence of infection in the field, there was actually almost 30% of the plants infected.

Field Trial No. 5

A further field trial was carried out using pot-grown plants which were grown outdoors and artificially inoculated at day 1.

At day 3, 50 organs (leaf/pod/spike) were sampled and placed in the apparatus of the invention and compared over a period of time with 50 organs which were still attached to the plants.

Disease development was scored at intervals on each leaf, as percentage organ area affected, and is presented below as mean area affected.

Day (time zero 0 day 3)

Crop Disease Detection System 5 8 10 13 16 20 (tissue)

Winter wheat cv. Septoria nodorum

Guar ian (glum-blotch) Field 0 0 0 0 2 5

(spike)

Invention 2 6 17 - - -

Jerusalem artichoke cv.Nahodka Field 0 0 3 9 16 24

(leaf) Powdery Mildew

Invention . 13 36 - - -

Spring oilseed Alt rnama brass_cae rape cv. Linetta (dark leaf and pod Field ⁰ 0 0 6 11 20 (pod) spot)

Invention 8 3, 64 - - -

1

Potato cv. Riytophthora infestans Kerr's Pink ( late potato blight) Field 0 0 0 .0 19 30 (leaf)

Invention 12 29 60 - - A further trial was carried out using the method and test apparatus according to the invention. The trial was based on inoculating samples of glasshouse grown material with five different diseases at day 0. The samples were analysed at time intervals for (a) average number of lesions per leaf, and (b) % of leaves infected for each disease. The results of the trial for each disease are plotted in Figs. 6 to 10.

These field trials demonstrate in a relatively simple manner the advantages of the present invention.

It is understood that an assessment of leaf area covered by a specific disease following incubation according to the method of the invention will be conducted by comparison with standard keys such as drawings showing 5%, 10%, 15% (by area) infection. An illustrated series of assessment keys for plant disease may be found in the paper by W.C. James in Canadian Plant Disease Survey __ (1971): 39-65.

Many variations on the specific embodiment of the invention will be readily apparent and accordingly the invention is not limited to the embodiment hereinbefore described which may be varied in construction and detail.

Claims

1. A method for the early detection of infection in a crop comprising the steps of:-

taking a sample from the crop;

detecting the presence of the accelerated infection in the crop sample.

2. A method as claimed in claim 1 wherein the control conditions include the step of retarding the senescence of the crop sample.

3. A method as claimed in claim 2 wherein the senescence of the crop sample is retarded by contacting the crop sample with a senescent retardant material.

4. A method as claimed in any of claims 1 to 3 in which the sample comprises one or more organ(s) detached from a growing crop.

5. A method as claimed in claim 4 in which the or each organ comprises a leaf.

6. A method as claimed in any of claims 1 to 5 wherein the development of infection in the crop sample is accelerated by incubating the crop sample under control conditions.

7. A method as claimed in any preceding claim wherein the development of infection in the crop sample is accelerated by illuminating the crop sample under control conditions .

8. A method as claimed in claim 7 in which the crop sample is illuminated for between 12 and 24 hours per 24 hour incubation period.

9. A method as claimed in any of claims 6 to 8 wherein the control conditions include temperature and humidity conditions .

10. A method as claimed in any of claims 3 to 9 wherein the senescence retardant material is in a liquid form.

11. A method as claimed in any of claims 3 to 10 wherein the senescent retardant liquid is of the type which substantially retards the growth of saprophytic fungi.

12. A method as claimed in any of claims 3 to 11 wherein the senescence retardant material is benzimidazole.

13. A method as claimed in any of claims 3 to 12 wherein the senescence retardant liquid is applied to the crop sample by contacting capillary material with the senescence retardant liquid and crop sample during acceleration of the development of infection.

14. A method as claimed in claim 13 wherein the senescence retardant liquid is applied to the sample by contacting capillary material with the senescence retardant liquid and crop sample substantially continuously during acceleration of the development of infection.

15. A method as claimed in any of claims 9 to 14 wherein the temperature is maintained between 10°C and 40°C for accelerating the development of infection.

16. A method as claimed in claim 15 wherein the temperature is maintained at between 16°C and 25°C.

17. A method as claimed in any of claims 9 to 16 wherein the humidity is maintained at predetermined levels during the acceleration of the development of infection.

18. A method as claimed in any preceding claim wherein a plurality of crop samples are taken and the development of infection in the crop samples is accelerated.

19. A method as claimed in claim 18 wherein the crop samples are divided into at least two cohorts and the development of infection in one cohort of crop samples is accelerated in different control conditions than the control conditions for accelerating the development of infection in another cohort.

20. A method as claimed in claim 19 wherein the development of infection in one cohort of crop samples in accelerated at relatively low humidity conditions and the development of infection in another cohort of crop samples is accelerated at relatively high humidity conditions.

21. A method as claimed in any preceding claim wherein the development of infection in the crop sample is accelerated in an incubator.

22. A method as claimed in any preceding claim including the step of assessing the level of infection in crop samples by:-

identifying the disease present;

calculating the number of samples infected;

calculating the area of each sample which is infected; and

recording the period before which infection symptoms appeared.

23. A method as claimed in any preceding claim including the steps of:-

comparing the level of accelerated infection in the crop sample against predetermined standards; and

assessing crop protection requirements accordingly.

24. A method for the early detection in a crop substantially as hereinbefore described with reference to the examples and drawings .

25. A method for protecting a crop against infection comprising the steps of:-

applying protection to the crop based on the early detection of the infection.

26. A method as claimed in claim 25 in which the protection comprises the application of a fungicide.

27. A method for testing a crop including the step of early detection of infection in the crop by a method as claimed in any preceding claim.

28. Test apparatus for carrying out the method for the early detection of infection in a crop as claimed in any preceding claim comprising an enclosure having;

a crop sample holder;

a sample holder mount; and

29. Test apparatus as claimed claim 28 wherein the enclosure is an incubator having a heater.

30. Test apparatus as claimed in claim 29 wherein the incubator includes a light source.

31. Test apparatus as claimed in any of claims 28 to 30 including a senescence retardant material holding and distribution means.

32. Test apparatus as claimed in claim 31 wherein the senescence retardant liquid holding and distribution means comprises a tray having a reservoir for senescence retardant liquid located below the sample holder mount, the sample holder, on mounting of a sample, projecting into the reservoir.

33. Test apparatus as claimed in claim 32 wherein the sample holder is constructed and arranged to receive a crop sample and a capillary material.

34. Test apparatus as claimed in claim 33 wherein the sample holder comprises a back plate and a front plate which are spaced-apart to receive the sample and capillary material therebetween, and clip means for the plates.

35. Test apparatus as claimed in claim 34 wherein the front plate has a recessed portion incorporating a plurality of inwardly projecting sample support dimples.

36. Test apparatus as claimed in claim 35 wherein the recessed portion of the front plate includes a plurality of through holes which are covered by a humidity control means.

37. Test apparatus as claimed in claim 36 wherein the humidity control means is a humidity controlling membrane.

38. Test apparatus substantially as hereinbefore described with reference to the accompanying drawings.