Milky Diseases of Beetles

Ira M. Hawley.

Grubs of the Japanese beetle live in the ground, where they may be attacked by several milky diseases, of which the type A, caused by Bacillus popilliae, is the most widespread and the most important.

That milky disease was first discovered in central New Jersey about 1933 when men who were conducting field surveys found a few abnormally white grubs. On microscopic examination, the late G. F. White, of the Bureau of Entomology and Plant Quarantine, perceived that the blood of the grubs was teeming with bacterial spores. The spores caused the white appearance and led to the designation milky disease for the ailing grubs.

The spores are spindle-shaped bodies about 1/4600 inch long so small that several billion may exist in one milky grub. When a healthy grub becomes infected with milky disease, the spores give rise to slender vegetative rods, which grow and multiply in the blood by repeated divisions and in a few days develop into the spore form.

As the disease develops, the blood of a sick grub, normally clear, becomes filled with the bacterial forms and milky in appearance. When affected grubs die, the spores, which had filled the body cavity, are left in the soil. They are taken up by other grubs as they feed on the roots of plants, and they in turn become diseased. As the process goes on the number of spores in the soil increases, more and more grubs are killed, and fewer beetles emerge.

In the rod stage the disease organism is comparatively short-lived, but the spores are long-lived. They resist excessive dryness or moisture, cold, and heat. They may remain alive in the soil for many years.

Many bacterial pathogens germs that cause disease of man, animals, and plants can be grown on artificial culture media and thus made to produce in great numbers the organisms that cause diseases. The culture possibilities of B. popilliae, which causes the type-A milky disease, have been intensively studied by S. R. Dutky, the bacteriologist of the Moorestown, N. J., laboratory of the Bureau of Entomology and Plant Quarantine, and by workers in State and private laboratories, but they have found no medium in which the rods will develop into spores.

The rods of type-A bacillus, however, will grow on several culture media and great numbers may be obtained. The rods may be transferred from one culture to another, where they will go on producing more. The milky disease may be started in healthy grubs by the injection of the rods into the blood stream with a hypodermic needle. Of the many ingredients for media tested in culture studies, thiamine and tryptophane have been found to be essential for growth and multiplication of the rod form. Other materials help somewhat. However, some element, which is required to bring about the change from rods to spores, has been wanting in all the many culture media we have tested. Work to find a medium in which spores may be obtained has been in progress since 1934.

Early in the study of the type-A milky disease we learned that it occurred largely in a small area where the Japanese beetle had been longest established in this country. In other words, the beetle had spread faster than the disease. We felt certain that the disease would help control the beetle in places where it did not occur if enough spores could be produced.

Because spores could not then ( and still cannot) be obtained in artificial culture media, Dr. Dutky developed a new technique for obtaining spores. In the process, the grub itself is used as the culture medium. Thousands of healthy grubs are dug in the field each fall and stored in cold cellars at the laboratory. They are removed as needed, and each grub is inoculated hypodermically with about 1 million type-A spores from milky grubs. The injected grubs are held individually at a temperature of 86 F. in cross-section boxes with soil and with sprouted grass seed as food for 10 to 12 days. In that time, each grub usually contains 2 billion or more spores. The United States letters patent covering the main features of the process were granted to Dr. Dutky, who assigned them to the Secretary of Agriculture.

Occasionally enough milky grubs are found in a field to justify collecting them for processing, but we seldom find a place with enough diseased grubs in the proper condition to pay to dig.

When the disease has developed to just the proper stage, the grubs are screened from the soil, washed to remove dirt particles, and placed in jars of ice water, which are stored in a refrigerator at about 35 F. The cooling quiets the grubs and prevents deterioration. When enough injected grubs have accumulated, the excess water in the jars is poured off and the grubs are ground up in a meat chopper. Samples are taken of the resulting mixture of spores and grub parts and are checked to see how many spores exist in each unit of the mixture. Enough chalk is added to standardize the mixture at i billion spores per gram. Then it is passed through a blower to break up the masses of particles. The mixture is dried by a blast of warm air and a filler, usually talc, is added to standardize the powder at 100 million spores per gram, roughly 2.8 billion spores per ounce. The powder is known as spore dust and it is ready for packaging.

If there were no loss in the making, 23 grubs containing 2 billion spores each would produce 1 pound of spore dust. Spore dust has been held in a dry condition for as long as 10 years without noticeable deterioration and it is always ready to use.

A program to colonize the disease in areas where it did not occur was started in 1939 by workers at the Moorestown laboratory in cooperation with State and Federal agencies. By the close of the 1950 season, more than 166,000 pounds of spore dust had been applied at nearly 122,000 colony sites. Nearly 93,000 acres had been treated in 199 counties in 14 Eastern States and the District of Columbia. At least 15,500 pounds of spore dust were used in treating properties owned or maintained by the Federal Government.

State entomological agencies have sometimes assisted in the production of spore dust by digging and inoculating grubs, which were then shipped under refrigeration to Moorestown, where all the processing into spore dust has been done. The spore dust from the grubs was then returned to the States for distribution. Records of the number of colonized sites in each county were supplied to the Moorestown laboratory. Workers at the University of Maryland have carried on a large-scale program of grub inoculation and distribution of spore dust since 1940.

BECAUSE SPORE DUST is difficult to make and costly, it is seldom applied to the soil as a complete coverage. In experimental applications it has been spread evenly over the ground with commercial fertilizer or some other filler and good disease infection has been obtained. There was no evidence that the fertilizer reduced the action of the disease spores. However, spore dust is usually distributed in spots with a modified rotary hand corn planter, which drops about 2 grams of the dust each time it is tripped. In treating a small yard, the powder may be applied in spots with a teaspoon. It is merely placed on top of the ground; the next rain will wash it in.

The amount needed for any area is regulated by the distance between the spots, which are usually 3 to 10 feet apart. When applied with a corn planter at a 3- by 3-foot rate, 2o.6 pounds of spore dust will be required to cover an acre. At a 5- by 5-foot interval, 7.0 pounds will be needed. When the spots are 10 by 10 feet, 1.75 pounds are required. The size of the treated tracts has varied greatly. Treatment of at least 2 half-acre plots per square mile is desirable in open agricultural country. In city areas, the treatment of the properties on at least 1 block in 10 is considered good. These rates of treatment are heavier than it has been possible to use in many places. Often the spore dust has been applied in only a few locations where grub counts were highest. In a few places where beetles have been present in destructive numbers, tracts of 100 acres or more have received a complete coverage with the spots close together. That was done to get a quick establishment of the disease.

Milky disease spores. A, vegetative rods; B, sporulating rods; C, mature spores.

In colonizing new areas, places are selected for treatment which have a permanent turf and a high count of grubs. Milky disease is not usually applied to fields that might be plowed or cultivated soon after treatment because the spores are scattered and buried before the disease can become established. Furthermore, the grub count is usually low in such places. The rapidity of disease build-up will depend on the number of grubs per square foot, the closeness of the spots in the treated area, and the number and size of treated tracts. Since grubs become diseased largely by feeding in infected soil, some time must elapse before the disease will spread over an entire field or larger area from the first grubs infected. Milky disease will be spread from those initial points by any natural or artificial movement of topsoil containing spores, by the movement of diseased grubs through the soil, and by birds and animals that feed on diseased grubs. It has been shown experimentally that live spores are present in the droppings of birds that have fed on diseased grubs. Spores of the type-A milky disease can withstand wide ranges of soil temperature and moisture conditions, but may lose some of their infective power after direct exposure to the rays of the sun for several days.