Appendix I: Wartime Studies into Potential Anti-Crop BW Agents This Appendix provides an overview of scientific research into the production and control of certain diseases against crops. The US BW research and develop- ment programme had made possible experimentation on pathogenic agents on a scale never previously envisaged. Whilst the US BW programme resulted in the attainment of military objectives in the field of BW, the programme also generated a considerable amount of information of relevance to the peace- ful use of pathogenic organisms. At this stage of the research and develop- ment phase it is only stated intent that distinguishes offensive research from defensive research.1 This investigation concerns factors that affect the infectivity of the pathogen which causes brown-spot of rice. According to Agrios:2 Helminthosporium diseases occur throughout the world and are very common and severe on many important crop plants of the grass family (Gramineae) and in some areas, they also cause diseases on apple (black pox) and on pear (blister canker). … Different species of Helminthosporium cause corn leaf blights, brown spot or blight of rice; crown and root rot of wheat, and the leaf spot of wheat; net blotch of barley, stripe disease of barley, and the spot blotch of barley; the Victoria blight and the leaf blotch of oats; eye spot and the brown stripe of sugarcane; and leaf spots or blights of turf grasses, and the crown and root rots (melting out) of turf grasses. The investigation involved research conducted under laboratory and field con- ditions at the US BW research and development facility at Camp Detrick, Frederick, Maryland; and at Beaumont, Texas, between April 1944, and October 1945. The causal agent of brown-spot of rice, Helminthosporium oryzae van Breda de Haan affects rice at each respective stage of development. Extensive damage is reported to occur in the seedling stage of development. In the post-seedling stage, further development is impaired by infection to the photosynthetic areas of the plant’s leaves, while during the mature stage of plant development, both the quality and the quantity of plant yield is affected adversely. While studies concerning the environmental factors affecting the infectivity of Helminthosporium oryzae have been undertaken, it is reported that such studies involved Oriental strains of the pathogen and that further investigation with US varieties are therefore justified. 217 218 Appendix I The investigation into the factors affecting the infectivity of Helminthosporium oryzae was organised into the following sub-sections: 1. materials and methods; 2. moisture relations; 3. air temperature; 4. time of day; 5. form of the pathogen; 6. effect of variety of host; 7. effect of age of host on susceptibility; and 8. discussion and summary. A summary of the above will be included in the paragraphs that follow. Materials and methods The following varieties of rice were selected for the purposes of the investiga- tion: Onsen and Butte. For experiments in the greenhouse, rice plants were grown in 0.5 gallon plant pots with 15 seedlings per pot. For experiments where it was necessary to grow plants into the mature stage of development, larger (one gallon) pots where used with five plants per pot. The following inoculum was used during the course of the experiments: a conidial-dust and vegetative mycelium3 dust. Moist sterilised oats or rye were utilised in the production of inoculum and 3–4 g. of conidia was produced from each respective 300-g dry substrate sample, with 96-7 per cent of the inoculum reported to be capable of germination. The process of producing conidial dust is described in the paragraph that follows: 4 Three hundred grams of seeds, placed in a Fernbach flask with 345ml. of water, were steamed for one-half hour, autoclaved for 20 min. at 15lb pres- sure, allowed to stand over night, and re-autoclaved. When the flasks had cooled, they were inoculated with an aqueous suspension of conidia and incubated at 22–24°C. in the dark for 14 days. Three to five 200-ml. portions of water were added singly to the flasks, vigorously shaken, and the resulting suspension of conidia removed from the substratum by straining through wire gauze into cylinders after each such addition. The cylinders were allowed to stand overnight in a room at 8°C. to allow the conidia to settle. The super- natant liquid was decanted, and the concentrated conidia were collected in a Buchner funnel on coarse filter paper. The filter cake was broken up and dried in a steam oven for 24hr. at 40°C. A mycelial dust of Helminthosporium oryzae was obtained by way the following process:5 Four-litre Pyrex serum storage bottles were used as culture vessels, with three litres of a medium composed of a solution of 10 percent (by volume) black- strap molasses and 0.5 percent peptone in each bottle. After sterilisation at 15 lb pressure for 20 min. and cooling, the bottles were inoculated with approximately two ml. of an aqueous suspension of conidia. During the incubation period of six days at 27–31°C. the cultures were aerated with sterile air introduced at a rate of 1500 ml./min. The mycelium was collected in a Buchner funnel, the filter cake broken up and dried at 40°C. for 24 hr. The resulting product was ground in a Wiley mill to produce a dust. Both conidia and the ground mycelium were mixed with a filler of limestone (500 mesh) immediately before use. Appendix I 219 A series of experiments was subsequently conducted using the above coni- dial/mycelial mixture, which was applied to rice plants with the aid of an improvised duster. Prior to this experiment the rice plants had been moistened with water dispensed from an atomiser. Plants inoculated in a cabinet, and out- doors, were then placed in a humidity tent where humidity was maintained at high levels. Three respective methods were adopted for evaluating the degree of infection. The first method took note of the number of lesions appearing per leaf in rela- tion to the known weight of inoculum used in the experiment, and the area exposed. The second method involved the microscopic examination of leaves in order to establish the relative number of conidia and lesions per leaf. The third method evaluated infection against three criteria: light infection; moderate infection; and heavy infection. The latter method was subsequently refined in order that the area of the infected leaves could be compared with a photographic standard that acted as a control. Moisture relations A series of experiments were then conducted in order to establish the effect of moisture upon infection, both prior to, and after inoculation. Three of the six plants used in this experiment were placed in a humidity tent for 24 hours prior to inoculation. The remaining three plants were not exposed to humidity. After inoculation both sets of plants were returned to the humidity tent for a period of 24 hours, then left to stand on a greenhouse bench. The experiment revealed, however, no difference in the degree of infection to either the control plants or the plants that had been exposed to humidity prior to inoculation. In order to establish the effect of water on plants at the time of infection the following experiment was conducted. Three separate sets of plants were used in this experiment. After being placed in a humidifier for a period of one hour, two sets of plants were thoroughly wetted with an atomiser and inoculated with conidial dust. One set was returned to the humidifier together with a third plant which had been inoculated when the leaves of the plant were completely dry. The remaining set, which had been exposed to humidity prior to inoculation, was placed on a greenhouse bench. After a period of three days no infection had occurred to the plant that had been placed on the greenhouse bench, and while infection was reported to be apparent on the plant that was inoculated with dry leaves and placed in the humidifier, infection was reported to be severe on the plant that had been exposed to moisture prior to and after inoculation. In a sub- sequent duplicate experiment conducted with mycelial dust, similar levels of infection is reported to have occurred. A further experiment estimated that optimum infectivity occurred at a temperature of 22°C after exposure to high humidity for a period of 10 hours. Two experiments were designed in order to determine the viability of conidia on leaves under greenhouse conditions. In the first experiment, by inoculating 22 pots of rice with conidia and then placing each pot in a humidity tent at intervals of 2 days, it was established that conidia remained viable for 18 days. In a subsequent experiment which extended the time period to 28 days it was reported that conidia remained viable for the period of the life of the leaf. In 220 Appendix I field experiments, where heavy rainfall occurred immediately after inoculation, it was found that conidia was washed from leaves reducing infection significantly. Air temperature An experiment was conducted in order to determine the influence of air tem- perature upon the severity of infection. Rice plants inoculated with conidial dust were placed in respective water baths at high moisture levels. The tempera- ture of water and air in the water baths was adjusted as follows, to: 15°C, 20°C, 25°C, 30°C, and 35°C. This test, and subsequent comparable tests with mycelium inoculum, suggested that the optimum range for infection was between 20°C and 25°C. Time of day In field testing conducted at Beaumont, Texas, an experiment was devised in order to establish the time of day that would result in the greatest amount of infection.