ZA200502817B - Cryptophlebia leucotreta granulovirus (CrleGV-SA) as a biological control agent - Google Patents

Description

Title of Invention ' CRYPTOPHLEBIA LEUCOTRETA GRANULOVIRUS (CrleGV-SA) AS A

BIOLOGICAL CONTROL AGENT

Description of Invention

Technical Field and Background Art

Cryptophlebia leucotreta is regarded as one of the most important pests of citrus and other fruit crops in southern Africa. Chemical control is problematic due mainly to the development of resistance and to biocontrol disruption. Cryptophelebia leucotreta granulovirus (CrleGV) is a baculovirus of the genus

Granulovirus that infects and kills Cryptophlebia leucotreta. A similar virus (CIGV-CV3) was described by Jehle et al (1992) and shown to infect and kill false codling moth (Fritsch, 1983).

Methods of rearing C. leucotreta have been described by Ripley ef al (1939), Theron (1948) and

Schwartz (1971). However, these methods involve the use of a Rhizopus sp. of fungus as part of the cycle. This is not suitable for the mass production of a biological control agent as the system as a whole is prone to large scale infection with Aspergillus spp. and is extremely labour intensive.

A biological control agent such as CrleGV has various significant advantages including the absence of a compulsory pre-harvest interval and the ability for CrieGV to be used with other biological agents to control C. leucotreta as part of an intergrated pest management system. CrleGV-SA, a novel South

African isolate of CrleGV, is a lethal virus of C. leucotreta. It has been developed as such an agent and is the subject of this patent.

Baxter ef al. 1999. Introductory Mycology Course Manual. Agricultural Research Council, Pretoria.

David. 1969. Journal of Invertebrate Pathology 14: 336-342.

Fritsch. 1988. Mitt. Dtsch. Ges. Allg. Angew. Ent 6: 280 - 283.

Jehle et al. 1992. Journal of General Virology 73: 1621-1626.

Jones. 2000. Bioassays of entomopathogenic viruses, In “Bioassay of entomopathogenic microbes and nemotodes (A. Navan & KRS Ascher, eds.) CAB Publishing, UK pp95-140.

Ripley et al. 1939. Science Bulletin of the department of Agriculture and Forestry of the Union of

South Africa No 207: 1-18.

Schwartz. 1971. Journal of the Entomological Society of southern Africa 34: 431-433.

Theron. 1948. Department van Landbou, Vrugtenavorsig Tegniese Reeks No 4 Wetenskoplike pamflet 262, 48pp.

Van Ark. 1995. Introduction to the analysis of quantal response. Agricultural Research Council

Agrimetrics Institute, Pretoria.

Disclosure of Invention ‘ Cryptophelebia leucotreta granulovirus

CrleGV-SA was isolated from infected C. leucotreta in South Africa and makes up part of this invention. It is a double stranded DNA granulovirus with a genome size by restriction fragment analysis of between 105 kilobases and 122 kilobases. The restriction enzyme digestion profiles of CrleGV-SA are shown in Table 1 and Drawing 1/6 (DNA profile of CrleGV-SA with six single restriction enzymes digests).

CrleGV-SA resembles most closely CIGV-CV3 of Jehle et al (1992) among the various granuloviruses isolated up to the present, but there are a number of distinct differences in the restriction enzyme digestion profiles that show that CrleGV-SA is a novel and unique granulovirus that infects and killsC. leucotreta.

Table 1 Size* of CrleGV-SA restriction fragments.

Fragment [EcoRI [Nael _ [Kpnl __ [BamAl _ [Xhol [Sad

B [82> [moa [2005 [23067 [21308 [27797

BD [78% [Bis [179m [9817 [115% [21458 £ [7534 [805 [i993 [isd

TF [75% [6%8 [1096 [89d [5041

ECHR AF SN SN TR IAS I

IL A FAY J XN AE RE v1 SN I 1 [som [4s [iss [sm 7 SN I SO A Ic I EE

K [aa [iss [ite7 fox

OS A A (A X11 SE A A

IS I A LF A 1 7 A I EE

N37 oes (ow

Ko A A I EA RE EN ER

EC J I I NE ER RE

KI XS ES NS I RE wees

EN 1 EN ES NR A I EE

I A 11 EO SA I I A v (ven 1 rr 1 1 v___ qvess | ~~! or 0]

I EIT RR PE EE EE

: BS XC I A A A v joes | 1 1

Zz |o4s *Expressed in kbp (kilobase pairs) relative to DNA molecular size standards.

Pathogenicity of CrleGV-SA against Neonate larvae of C. leucotreta , Surface Dose-response

L.Cso (concentration required to kill 50% of larvae in a amps) and L.Cy (concentration required to kill ) 90% of larvae in a sample) were estimated to be 3.903 x 10° OBs (occlusion bodies)/ml and 5.472 x 10°

OBs/m] respectively.

Surface Time-response

LTsp and LTyo were estimated to be 4 days 5 h and 6 days 10 h, respectively.

Pathogenicity of CrleGV-SA agaimst Fifth instar larvae of C. leucotreta

Surface Dose-response

LCs and LCgy were estimated to be 1.724 x 10” OBs/ml and 2.279 x 10° OBs/ml respectively

Surface Time-response

LTso and LTyo were estimated to be 8 days 4 hand 9 days 18 h, respectively.

Thus, CrleGV-SA shows significant pathogenicity against C. leucotreta larvae both in the neonate and 5" instar developmental stages.

Modes for carrying out the invention

The method of host rearing

The C. leucotreta larval culture is kept at approximately 27°C and 30% relative humidity. Moths are held at the same temperature and approximately 60% humidity.

Emergence and oviposition unit

One unit consisting of 10 eclosion compartments was designed and constructed. Pupae from different dates were placed into each compartment. Oviposition cages were attached to each compartment (Drawing 2/6: Incorporated moth eclosion and oviposition unit for C. leucotreta. The oviposition cage on the top right is not covered with netting so that the wax paper conveyer belt system can be demonstrated.). The oviposition cage was made with a thin wire frame, covered with organdy netting.

Wax paper, on a roll, was fed into the oviposition cage through a thin slit at the bottom of the cage. The paper covered the floor of the cage and exited through a similar slit on the opposite side of the cage. : Every 24 hours the wax paper, with eggs now deposited on it, was moved on by the length of the cage and cut. The eggs were used either for further production of C. leucotreta or production of egg parasitoids.

Because of the thin wire framework, there was very little alternative surface area on which moths could oviposit. Sieve rims (used in the original method for rearing the moth) probably provided a greater . alternative surface area than did the framework of the new oviposition cages. Consequently, the number of eggs produced in the new oviposition cage per equivalent number of moths was 9.3% higher than produced in the sieves. The new oviposition cage was therefore an improvement on the previously . used oviposition system.

Contamination reduction

A significant improvement has been made in reducing the level, and frequency of occurrence, of contamination in the larval diet, both by treatment of the diet and of the host eggs placed onto the diet.

Dipping eggs into a 0.15% solution of Sporekill (active ingredient: poly dimethyl ammonium chloride 120 g/L; Hygrotech International, South Africa) for 15 minutes was a highly effective and safe surface sterilisation treatment. The details of the treatment were deduced after testing a range of concentrations (of Sporekill) and exposure times (Table 2). Formalin had previously proved both unsatisfactorily effective and detrimental to egg survival.

Table 2 Effectiveness of Sporekill for surface sterilisation of C. leucotreta eggs and its effect on egg survival. lium contamination [Unireated | ®857az267 [010 [10 (0 [04% Sporek dip (55) | 76.82a2553 [0 [100 [10] [0.2% Sporekill dip (1 mim) | 85.83a24.89 [0 [0 [0 Jo [02% Sporekill dip (4 min) | 75.46a2328 [0 [9 9 JW0 [0.1% Sporekildip (15 min) [811923636 [0 [3 [013 [0.1% Sporekill dip Amin) [86822442 [0 [4 — [2 [6 *Values in the same column followed by the same letter are not significantly different (P<0.05; Bonferroni multiple range test).

Both nipagin (p-Hydroxybenzoic acid methyl ester) and sorbic acid were added to the diet to further control contamination. The ideal concentration of nipagin, to control fungal growth and not impede C. leucotreta development, appeared to be somewhere between the two lowest rates tested (Tables 3). The addition of sorbic acid to the diet aided in the further suppression of fungal contaminants without a significant increase in moth mortality (Table 4).

Table 3 Effect of different concentrations of nipagin on fungal growth and C. leucotreta development, when added to the diet.

Standard Rhizopus sp. diet 16.5abc + 1.5 Vigorous

Nipagin 0.2 g/jar 30.0a+ 11.0 Suppressed

Nipagin 0.4 g/jar 7.5bcd £ 1.5 None

Nipagin 0.6 g/jar 6.0cd + 3.0 None

Nipagin 0.8 g/jar 20d+£1.0 None

Nipagin 1.2 g/jar 1.0d £0 None oo in the same column followed by the same letter are not significantly different (P<0.05; Bonferroni multiple range test).

Table 4 Effect of different concentrations of sorbic acid on fungal growth and C. leucotreta development, when added to the new diet**.

Treatment Moths produced/jar* | Fungal Proportion (%)

Eee EET (8 jars/treatment) DAT*** fungal contamination 26 DAT***

New diet (without sorbic acid)** 63.9a + 5.1 Superficial. 78.1b£3.9

Plus 0.10 g sorbic acid/jar 69.1a+ 8.0 Clean. 63.7b £10.5

Plus 0.13 g sorbic acid/jar 53.6a+8.5 Clean. 44a+26

Plus 0.17 g sorbic acid/jar (no | 72.6a+ 7.0 Superficial. 75.6b+3.9 nipagin) *Values in the same column followed by the same letter are not significantly different (P<0.05; Bonferroni multiple range test). ede Table 5 for recipe. *#*DAT = days after treatment.

With the addition of anti-microbial agents (nipagin and sorbic acid) to the diet, the use of Rhizopus sp. fungus as a source of nutrition became impossible. 1t therefore became necessary to add various nutritional ingredients to the diet. An artificial diet for C. leucotreta, which does not require the use of

Rhizopus sp., and which consistently produces good numbers of moths has been developed, (Table 5).

Table 5 Recipe of new artificial diet.

Maize meal 2000 g 40 g

Wheat germ 200g 4g

Casein (later replaced with milk powder*) 365g 073g . Brewer's yeast 100 g 2g

Nipagin 15g 03g ; Distilled water 800 ml 40 ml *Nestlé® Nespray Instant Milk Powder.

Production of pupae on the new diet was comparable to that on the old standard Rhizopussp. inoculated diet (Table 6). However, pupation on the new diet was delayed by four days (Table 6). This would not . be a problem in a continuous production system. Egg laying of the F1 generation was 19.83% higher when reared on the new diet compared to the old diet (per equivalent number of moths) (Table 6). A further observation in favour of the new diet, was that there appeared to be far greater synchrony of . development. Nutritional value of the new diet is obviously similar throughout the medium, whereas proximity to the Rhizopus sp. growth affects nutritional value in the old diet. This results in a conspicuous spread of life stages in the Rhizopus sp. diet. Greater synchrony of life stages is certainly desirable for efficient and organised mass rearing.

Table 6 Development of C. leucotreta on a new artificial diet relative to the standard Rhizopus sp. inoculated diet.

Development Duration of | Eggs/240 | Contamination (pupae/jar)* development | moths (evaluated 16 days after (20 jars/treatment)** | from egg to introducing eggs onto pupa (days) diet)

Rhizopus sp. 34.35a+ 1.6 18882 Aspergillus growth ee ES Rl vcr i)

New artificial 33.10ax 1.7 21 22626 Aspergillus growth ol A Gl rv * Values in the same column followed by the same letter are not significantly different (P<0.05; Students’ T-test). ** Average of 67 eggs placed into each jar for each treatment.

Method of virus production

CrleGV is mass produced in 220 ml glass pie dishes. The artificial diet (94 g dry ingredients with 100 g distilled water) is prepared in the same manner as previously described. The only difference between the diet used for virus production and the diet used for host rearing is that 33% higher concentrations of the anti-microbial agents (nipagin and sorbic acid) are used in the virus production diet.

Once cooled, the surface of the diet is inoculated with 14 ml of the calculated LCy of CrleGV for fifth instar larvae (2.279 x 10° OBs/ml) — adjusted to give the correct dose/mm? of diet. LCgo was calculated from inoculating diet with 200 pl of virus per 400 mm? (surface area of diet in one cell of a bioassay tray). This translates to a volume of 19 ml of a solution of L.Cyg to inoculate the surface of a pie dish of diet (38013 mm?) for virus production. However, such a volume was clearly far more than needed to thoroughly wet the diet surface. It was determined that 14 ml was sufficient to adequately and evenly wet the diet surface in a pie dish. To ensure that the surface of the diet is inoculated with this number of

OBs, it is sprayed with 14 ml of 3.093 x 10° OBs/ml. (Correct volumes and concentrations of purified virus are aliquotted and stored at -40°C). This is applied as a fine spray using a 1 £ plastic spray bottle.

The inoculated diet is placed in a laminar flow cabinet for +30 minutes, to dry. , A total of 300 fifth instar larvae are placed on the diet surface. The pyrex dish is covered with a double layer of “Gladwrap” (plastic stretch film) to prevent larvae from escaping. : Method of virus harvesting

Inspections are conducted once or twice a day, and any larvae showing symptoms of viral infection ate collected and stored at -40°C,

A higher recovery of infected larvae relative to introduced larvae was obtained with 300 larvae per container than with any other number tested (Table 7). Increasing the density per container by 33% (to 400) seemed to detrimentally affect the harvest of virus infected larvae. With increasing density, larvae also appeared to die more rapidly. The greater density probably caused this in two ways. Firstly, it increased the opportunity for horizontal transmission of the virus and therefore larvae were ingesting greater quantities of virus. Secondly, a greater level of stress could have been experienced by the larvae due to “overcrowding” which in turn caused a manifestation of the usually nomapparent level of infection (which was known to be present in the C. leucotreta culture) on top of the induced epizootic.

Table 7 Harvest of virus infected larvae from diet inoculated with the LCs (2.279 x 10 OBs/ml).

Larvae Replicates Larvae Harvest (%) | Period of | Peak harvest introduced harvested harvesting (DAT) (ave.) (DAT?) 200 5 62.2 31.1 7-15 9.2 250 4 121.7 487 6-12 8.5 300 4 167.0 56.0 6-11 8.5 400 3 154.0 38.5 3-9 7.0 : *DAT = days after treatment.

Only 43.1% of all larvae introduced onto the diet were eventually harvested as virus infected larvae (Table 7). This was unacceptably low, although the harvest of 56.0% when 300 larvae were placed onto inoculated diet, was somewhat better.

Harvesting of the entire production dish — larvae and diet, was also investigated, as the harvesting of larvae individually was labour intensive, and recovery rate of larvae (i.e. number of infected larvae harvested per number of healthy larvae placed on inoculated diet) was considered poor. Three pie dishes of diet were prepared and inoculated. Three hundred fifth instar larvae were placed into each dish. After eight, nine and 10 days the entire contents of one pie dish was harvested and frozen at- 40°C.

Semi-purification of virus from diet

Larvae (or larvae and diet) are defrosted and virus liberated either by crushing with a mortar and pestle or homogenising in a Dupont omni-mixer. The homogenate is then filtered through a double layer of muslin cloth, to produce a crude viral suspension. The suspension is diluted 1:4 with 0.1% SDS in distilled water and centrifuged at 1500 rpm (using a JA-20 Beckman rotor) for 2 minutes, to pellet insect debris. The supernatant is retained and the pellet resuspended in a few millilitres of 0.1% SDS. This is centrifuged again in the same manner and the resulting supernatant again retained. The process is repeated and all retained supernatant added together. The pellet is discarded.

The supernatant is then spun at 9000 rpm (using a JA-20 Beckman rotor) for 30 minutes. The resulting pellet consists of two layers: a dark lower layer (virus) and a pale upper layer. While the darker layer is retained at 4°C in a few millilitres of 0.1% SDS, the lighter layer is resuspended in 0.1% SDS by vortexing. This is centrifuged at 15000 rpm in a desktop microfuge for 10 minutes. Again the pellet consists of two layers. The top lighter layer is discarded. The bottom darker layer is added to the virus layer previously retained in 0.1% SDS and either refrigerated or frozen.

For application against C. leucotreta in the field it is not necessary to do any more than semi-purify the virus, at most. This is actually preferable to purified virus, as the spray suspension would then contain more small particles which could offer protection to the virus against harmful ultra-violet (UV) rays.

Simple filtration of the initial homogenate (of infected larvae or larvae and diet) may even be sufficient for use in field trials. However, semi-purification is necessary and sufficient tobe able to enumerate the virus using a counting chamber (Jones, 2000).

Table 8 Yield of CrleGV by in vivo production in fifth instar C. leucotreta larvae and harvesting of larvae individually.

Number of | Total Mass Total OBs Mean OBs per | Mean OBs per

CrleGV infected | Mass per larva (larval | gram larval cadavers larva equivalent) 993g |0053g |3.731x10% [9.918x10° 1.872 x 102 1291 6624g |0051g [9.815x10" 7.603 x 10° 1.482 x 10” 827g |0046g |2436x10% |6.136x 10° 1.333 x 10" 1121 5358 |0.048g |3.412x10" 3.043 x 10° 6.367 x 10"° 7906 382602 |0.048g |3.318x10" 4.196 x 10" 8.672 x 10"

Table 9 CrleGV yield from harvesting larvae with diet (300 larvae per treatment).

Time of harvesting | Number of OBs| Number of OBs per| Number (and%) of (days after inoculation) harvested larva (300 larvae) pupating larvae 9 5.086 x 10"? 1.695 x 10" 8 (2.7%) 5.280 x 10" 1.760 x 10'° 4 (1.3%)

Yield of CrleGV (per larva and per dish), when larvae and diet were harvested together (Table 9) was lower than when larvae were harvested individually (Table 8). However, the labour saving involved (and consequently the cost saving) in harvesting the entire contents of the production dish ought to be . substantial when producing virus on a commercial scale. In addition to the labour saving, a further benefit of harvesting the diet with the larvae, is that the fine diet particles, if present in the final viral preparation for field use, could offer some protection against UV inactivation. Such a preparation will ) have to be well formulated so as not to block spray nozzles.

Microbial contamination

A comparison of the level of microbial contamination in five different preparations was conducted. . These were virus purified by glycerol gradient centrifugation, and four batches of semi-purified virus: from larvae individually harvested when symptomatically infected and from larvae harvested with their diet after eight, nine and 10 days. Three dilutions of each treatment were made in a 10-fold series: 10X, 100X, 1000X, 10000X, 100000X and 1000000X dilutions in sterile distilled water. Eight droplets (110 ul) of each dilution were placed in circular formation onto a PDA (potato dextrose agar) plate (plastic petri dish) (Baxter ef al., 1999). Droplets did not merge together and did not touch the edge of the container. This was done within a laminar flow cabinet. Once the droplets had dried, the lids were placed onto the plastic petri dishes and they were incubated at 27°C for three days. After two days the number of bacterial colony forming units (CFUs) was counted. After three days number of droplet points with fungal growth were counted. No attempt was made to identify the dominant bacterial species present.

The CFU:OB ratio in all of the semi-purified preparations was favourably low and substantially lower than in the purified preparations (Table 10). The lowest level of contamination, both bacterial and fungal, occurred in the semi-purified preparation of larvae and diet, which was harvested eight days after inoculation and this is the preferred embodiment of the invention

Table 10 Microbial contamination of different preparations of CrleGV.

Harvested Purification CFUs/ml CFUs/larva | CFU : OB | Fungal contamination per ml*

Glycerol gradient | » 354 106 | 6.349 x 10° | 1:4 793 4.545 x 10° centrifugation 3.182x 107 {2.014 x 10° | 1:208342 [9.090 x 10°

Larvae and diet: § days after | Semi-purification | 6.445x 10° | 4.608 x 10° | 1:433594 | 9.090 x 10' inoculation

Larvae and diet: 9 days after | Semi-purification | 1.164 x 10° [8.323x10* | 1:203 652 3.636 x 10* inoculation

Larvae and diet: days after | Semi-purification | 1.248 x 10° [9.988 x 10° | 1:176211 |6.363 x 10° inoculation *Fungal contamination was defined as the number of droplets, out of the total of eight, which had fungal contamination (at the highest dilution at which fewer than eight droplets were contaminated) per millilitre of the original preparation.

Industrial Applicability : Persistence of virus

A high density orchard (1664 trees planted per ha) of 10 year old Delta Valencias on the Qutspan

Foundation Block outside Uitenhage (33° 45'S, 25° 24' E), Eastern Cape Province, was selected for this trial. On 21 August 2001 seven trees were each sprayed with 17 £ of a crude CrleGV concentration of

7.67 x 10" OBs/ml using a 1000 £ capacity high pressure spray machine (made by Janisch, South Africa) with hand held spray guns. Pressure was set at 20 bars and 2 mm diameter nozzles were used in the guns. This rate of application would translate into a rate of 2.17 x 10'° OBs/ha. The application was more thorough than would normally be necessary; however, it was important to ensure absolute coverage of the fruit for the purpose of this trial. Daily weather data for the Uitenhage area were “provided by the South African Weather Service. This has been summarised in Table 11.

Table 11 Summarised weather data, obtained from the South African Weather Service, for the

Uitenhage area from 21% August 2001 to the 11" September 2001.

Temp. Sunshine time Rainfall Wind speed Wind direction (°C) | (h : min) (mm) (km/h)

At 0 days (immediately after the spray had dried), 1 day, 3 days, 6 days, 7 days, 14 days and 21 days after treatment, 30 fruit were picked from the outer northern (sunny) side of sprayed trees, 30 fruit from the inner southern (shady) side of sprayed trees, and 60 fruit from unsprayed trees. Thirty of the unsprayed fruit were dipped in a CrleGV concentration of 7.67 x 10’ OBs/ml in the laboratory and allowed to dry on a wire mesh drying rack. The other 30 fruit were left untreated. Two neonate larvae were placed onto each fruit, using a size 000 paint brush. A separate brush was used for each treatment and all brushes were sterilised in 2% sodium hypochlorite and rinsed in sterile distilled water before use.

Fruit were kept at 27°C and were inspected and dissected 14 days after the larvae were placed onto them. Number of fruit infested was recorded.

Reduction in infestation of the three treatments was determined relative to the infestation in the control fruit (Drawing 3/6: Reduction in C. leucotreta infestation of fruit (Delta Valencia oranges), relative to untreated fruit (0% on y-axis), when treated with CrleGV on the northern aspect of trees(” = ~ =), on the southern aspect of trees (———) and in the laboratory (——).).

Half-life of the virus was estimated by conducting probit analysis (Finney, 1971) on data measuring the reduction in infestation relative to infestation of untreated fruit using PROBAN (Van Ark, 1995). The equation for the line fitted to the data for fruit on the northern aspect of trees is y = 7.2561 - 1.0851x and the standard error of the slope is 0.3728 (Drawing 3/6). Half-life of the virus on the northern side of trees was estimated from the probit line to be 5.0 days.

The equation for the line fitted to the data for fruit on the southern aspect of trees is y = 6.6118 -0.4868x and the standard error of the slope is 0.2062. Half-life of the virus on the southern aspect of trees was estimated from the probit line to be 85.86 days. This is undoubtedly an inaccurate estimate, as at no stage up to 21 days of field weathering, did the infestation in the southern aspect treated fruit, increase to above 50 % of the infestation in untreated fruit. This trial will therefore have to be repeated and continued, at least until efficacy of the virus in the field is reduced by 50 %, in order to obtain a reliable estimate of half-life. However, it can be stated with fair certainty that the half-life exceeded 21 days.

The trial was applied in August, which falls during the South African winter. Average sunshine hours per day are obviously less than during summer, when most of the sprays against C. leucotretq wil) be applied. It can be expected that half-life in the summer months will probably be shorter.

In order to avoid immediate and substantial inactivation of the virus, applications should be made in the evening. Not only does this allow neonate larvae to ingest unaffected virus for several hours until sunrise the following morning, but wet virus has been found to inactivate more rapidly than dry virus (David, 1969). In this trial CrleGV was applied at 16h00, shortly before the sun set.

Because of the difference in rate of breakdown between virus on the northern aspect and virus on the southern aspect, control strategies with CrleGV could be influenced. It may be necessary to spray the northern side of trees more frequently than the southern side, or to include a UV protectant in tank mixes applied to the northern side, but not to the southern side.

Efficacy of virus

During 2001 two field trials were conducted to test the efficacy of CrleGV against C. leucotreta on citrus. In both trials, sprays were applied using the same spray machine (and hand guns) at the same settings as described for the persistence trial. All trials were laid out as single tree treatments replicated times in a randomised block design. Order of treatments within blocks was determined using random number tables. Application of treatments was commenced shortly after pheromone traps indicated an increase in moth activity. Bladbuff 5, at a concentration of 75 ml/100£, was added to the tank for all CrleGV treatments, in order to improve wetting and spreading. Spraying was always conducted from late afternoon to evening (between 15h00 and 19h00) so as to avoid immediate UV inactivation of the virus. Alsystin (benzoylated urea: triflumuron), an insect growth regulator (chitin synthesis inhibitor), was included in trials as a chemical standard.

A week before evaluation of trials began (which was three weeks after application), fruit lying on the ground underneath the trees were removed. Each week thereafter, fruit dropped from each tree were collected in paper bags, which were labelled with the treatment (name or number). This was continued either until fruit were harvested or there was no longer any difference between treatments. Fruit were inspected and dissected in the laboratory, and the cause of drop recorded. Before inspection, the labelled side of the bag was faced away from the inspector. Only once the inspection results for the fruit from a particular bag had been recorded, was the label noted. This was done to avoid any bias in the inspection process.

The first trial was conducted on mature Palmer navel orange trees on Sun Orange Farm near Addo (33° 34'S, 25° 40’ E) in the Sundays River Valley, Eastern Cape Province. Again two CrleGV treatments (8.31 x 10° OBs/m! at a rate of 1.22 x 10" OBs/ha and 7.46 x 10’ OBs/ml at a rate of 1.01 x 10'S

OBs/ha) and Alsystin (10 ml/100 £) were applied. An average of 38.3 £ of spray mix was applied per tree. Sprays were applied between 15h00 and 17h30 on S April 2001. Fruit drop was evaluated from three weeks (21 days) to nine weeks and five days (68 days) after application.

At Sun Orange Farm, from three weeks after treatment until fruit were harvested at nine weeks and five : days after treatment, C. leucotreta infestation was lower for all three treatments than the untreated control (Drawing 4/6: Weekly C. leucotreta infested fruit (Palmer navel oranges) drop from untreated control trees (white bars), Alsystin treated trees (grey bars), trees treated with 8.31 x 10° OBs/ml : CrleGV (first of two black bars) and trees treated with 7.46 x 10’ OBs/ml (second of two black bars), at

Sun Orange Farm, Eastern Cape Province, on 5 April 2001. Bars per week with the same letter are not

I significantly different (P<0.01; Bonferroni multiple range test). (*8.4 = 8 weeks and 4 days; *9.5=9 weeks and 5 days).). During no particular week were these differences significant, probably a reflection : on the relatively small number of replicates (i.e. 10 per treatment). During weeks three and four after application, reduction in infestation for the CrleGV treatments was greater than for the Alsystin treatment (Drawing 4/6). It is understandable that the efficacy of CrleGV would become apparent . before that of Alsystin, as Alsystin is an ovicide, only effective against eggs that are laid on an existing residue, and CrleGV is a larvicide. There would therefore be an apparent time delay in the working of

Alsystin, particularly if there were numerous C. leucotreta eggs laid before spraying. CrleGV would, however, be immediately effective against any neonate larvae. Despite the difference in mode of activity, it is surprising that a notable reduction in infestation of fallen fruit from Alsystin treated trees, was recorded only from six weeks after application. Thereafter, C. leucotreta infestation in Alsystin treated fruit was lower than in CrleGV treated fruit.

The sudden rise in C. leucotreta infestation in CrleGV treated fruit, from five weeks after treatment and the higher level of infestation than in Alsystin treated fruit from the following week, may have been caused by the rainfall during the first three weeks after application (80.3 mm). Rainfall was particularly high during the second week after application (50 mm). It is possible that CrleGV is not as rainfast as has been reported for other baculoviruses on other crops

Until six weeks (42 days) after treatment, there was little difference in infestation of fruit treated with the two different concentrations of CrleGV. From 42 - 60 days after treatment, C. leucotreta infestation was lower in fruit from trees treated with the higher concentration of CrleGV (Drawing 4/6). In the final evaluation at 68 days (9 weeks and S days) after treatment, there was little difference in fruit infestation between the CrleGV treatments and the untreated control. If this was indicative of a breakdown in the efficacy of the CrleGV treatments, and if infested fruit took three weeks (21 days) to fall, then CrleGV had protected the fruit for less than 47 days. If a breakdown in efficacy could have been recorded immediately after the penultimate evaluation (8 weeks and 4 days after treatment) then the fruit would have been protected for 39 days.

If fruit infestation per tree per week was averaged over a period of 21 - 60 days, all treatments significantly reduced infestation (Drawing 5/6: Mean C. leucotreta infested fruit (Palmer navel oranges) drop over the period, 21 - 60 days after application, for treatments applied at Sun Orange Farm, Eastern

Cape Province, on 5 April 2001. Arrows indicate reduction in infestation relative to untreated trees.

Bars with the same letter are not significantly different (P<0.01; Bonferroni multiple range test). (CrleGV 1X = 8.31 x 10° OBs/ml; CrleGV 10X = 7.46 x 10” OBs/ml). Infestation was 58.5% lower in

Alsystin treated fruit and 45.1% and 59.8% lower for the two CrleGV treatments.

Table 12 Estimated average production and sale of navel oranges (Shaun Brown, Capespan, personal communication).

Export Export Gross Cost/carton Net income | Mean Net volume/ha cartons/ha income/ | (production, per carton | number of | value/ carton) shipping)

During the period that the trial at Sun Orange was monitored, the untreated control lost around one fruit per tree per week to C. leucotreta. 1f CrleGV reduced fruit infestation by 59.8% (Drawing 5/6) for 39 : days, and there are 555 trees per hectare (the most common modern planting spacing), then each application of CrleGV could prevent a loss of | 849 fruit. If the average profit value of a navel orange is R0O.21 (Table 12) then this would be a saving of R388.31 per hectare. For an application of CrleGV ’ to have been justified in this situation, it would have had to cost less than R388.31 per hectare.

Obviously, the greater the number of fruit that can be saved by a CrleGV application, the more easily justifiable is any cost. It will, however, be important to determine what level of activity/infestation is too high for CrleGV to be acceptably effective. Another factor that a CrleGV application will have to be measured against, is the cost of applying Alsystin. Each application of Alsystin (10 ml/100£), if applied adequately on mature orange trees, would cost in excess of R2 000 per hectare. This is extremely expensive.

If CrleGV is applied sufficiently close to harvest so as to be able to reduce the chance of undetectable infestation in harvested fruit occurring, the cost of spraying is far more easily justified. If infested fruit are packed, fruit can decay en route to the market causing more dire and costly losses. Fungal infection in one decaying fruit can spread rapidly and easily to the other fruit in the carton.

The second trial was laid out as single-tree treatments replicated 10 times in a randomised block design at Rietfontein Farm. Order of treatments within blocks was determined using random number tables.

Treatments were applied on 11 April 2001. The two concentrations of CrleGV used were 7.02 x 1¢

OBs/ml at a rate of 1.15 x 10'* OBs/ha and 7.02 x 10” OBs/ml at a rate of 1.07 x 10" OBs/ha. CrleGV treatments were compared with an Alsystin (10 mi/100 £) spray, as the chemical standard, and with untreated control trees. An average of 31.5 £ was applied per tree between 15h00 and 17h00.

Infestation of dropped fruit was evaluated weekly from three to six weeks after application. The grower was repeatedly requested not to remove fruit from beneath trial trees. Despite this, before evaluations could be conducted on weeks four and six (after treatment), the grower collected and removed fruit from the trial block, whilst performing orchard sanitation. Fruit infestation results were therefore only available for the third and fifth weeks after application. This data was pooled and reduction in fruit infestation was calculated. Alsystin reduced infestation by 15.4%, and the two concentrations of

CrleGV by 61.5% and 46.1% (Drawing 6/6: Mean infestation in dropped fruit (Palmer navel oranges) per tree for a period of 3 - 5 weeks after treatment, for treatments applied for control of C. leucotreta at

Rietfontein Farm, Eastern Cape Province, on 11 April 2001. Arrows indicate reduction in infestation relative to untreated trees.). Similarities were noted in trends between this trial and the one conducted at Sun Orange Farm. Firstly, very little reduction in infestation could be detected for Alsystin up to and including five weeks post treatment. Secondly, the higher concentration of CrleGV did not provide a greater reduction in infestation than did the lower concentration, up to and including five weeks after treatment. In fact, in both trials, the inverse was true.

Claims (20)

Claims:

1. A biological control agent for Cryptophlebia leucotreta comprising a biologically pure culture of a double-stranded DNA granulovirus, having an average total genome size of at least 99% similarity to the average total genome size of the virus of which the restriction enzyme digestion profile is set out in Table 1, when subjected to restriction endonuclease analysis with EcoRl, Ndel, Kpnl, BamHI, Xhol and Sacl.

2. The biological control agent as claimed in claim 1, for use on agricultural Crops.

3. The biological control agent as claimed in claim 2, wherein the agricultural crops are agricultural fruit crops.

4. The biological control agent as claimed in claim 3, wherein the agricultural fruit crops belong to the genus Citrus.

5. The biological control agent as claimed in any one of the preceding claims for use, together with any other agent, as an integrated pest management system, the other agent being selected from the group consisting of biological agents, pathological agents, pharmacological agents and chemical agents. 14 AMENDED SHEE

14A

6. The biological control agent as claimed any ore of the preceding claims, having pathogenicity against larvae.

7. The biological control agent as claimed in claim 6, wherein the larvae are neonates.

8. The biological control agent as claimed in claim 6, wherein the larvae are at the fifth instar developmental stage.

9. The biological control agent as claimed in any one of the preceding claims, for use in the manufacture of a spray comprising items selected from the group consisting of UV protectants and a wetting and spreading agent.

10. An artificial diet suitable for use in cultivating the biological control agent of claim 1, including ingredients selected from the group consisting of maize meal, wheat germ, casein, brewer’s yeast, anti-microbial agents and distilled water.

11. The artificial diet as claimed in claim 10, wherein the ratio of dry to wet ingredients is 47:50. 14A AMENDED SHEET

12. The artificial diet as claimed in claim 10, wherein the anti-microbial agents are selected from the group consisting of methyl p-hydroxybenzoate and sorbic acid.

13. The artificial diet as claimed in any one of claims 10 to 12, for use in mass production of the biological control agent of claims 1 to 9.

14. The artificial diet as claimed in any one of claims 10 to 12, for use in virus production.

15. The artificial diet as claimed in any one of claims 10 to 12, for use in host rearing.

16. A biological control agent for Cryptophlebia leucotreta comprising a semi- purified culture of a double-stranded DNA granulovirus, having an average total genome size of at least 99% similarity to the average total genome size of the virus of which the restriction enzyme digestion profile is set out in Table 1, when subjected to restriction endonuclease analysis with EcoRI, Ndel, Kpnl, BamHI, Xhol and Sacl.

17. An oviposition unit, suitable for use in host rearing the biological control agent as claimed in claim 1, comprising an enclosure having insect-proof mesh, the enclosure having at least two opposing openings located towards 14B AMENDED SHEET

14C the lower half of the enclosure, so that in use, host organisms are forced to oviposit on wax paper inserted into the cage and located on the floor by feeding the wax paper through the openings, thereby increasing the yield of oviposition.

18. A biological control agent as claimed in claim 1, substantially as herein described and exemplified, with reference to the accompanying figures.

19. An artificial agent as claimed in claim 11, substantially as herein described and exemplified with reference to the accompanying figures.

20. An oviposition unit as claimed in claim 18, substantially as herein described and exemplified with reference to the accompanying figures. 14C AMENDED) Geis +.