Disease of Silkworms – White Muscardine

One of the most common fungal parasitic silkworm diseases is caused by Beauveria bassiana invading the silkworm body through the skin. The disease is named after the diseased silkworm carcass dried up and covered with white conidia. Beauveria bassiana is widely distributed, especially in warm and humid areas. It can cause damage regardless of the silkworm stages of spring, summer and autumn.

In China, “Shen Nong’s Materia Medica” has a record of “white dead silkworm, taste salty”. In the Song Dynasty “Chen Yangnong Book · Sericulture” (1149), there is a description of “…the cold wind is the black and white red stiff”. In 1835, the Italian botanist Agottino Bassi used experiments to prove that Beauveria bassiana was an infectious silkworm disease caused by a fungus parasitic. Around the 1950s, silk producing countries such as China and Japan experimented and promoted bleaching powder anti-stiff powder, Xilisheng anti-stiff powder and Sailisan anti-stiff powder and other agents, which effectively controlled the harm of Beauveria bassiana.

The pathogen Beauveria bassiana belongs to the genus Beauveria bassiana, the scientific name is Beauveria bassiana (Balsamo) Vuillemin, and it was once called Botrytis bassiana. The growth and development cycle of Beauveria bassiana is divided into three stages: conidia, vegetative hypha and aerial hypha. The propagules are conidia, colorless, spherical or ovoid, with a length and width of 2.5 to 4.5 × 2.3 to 4.0 microns, and the conidia appear white when they are gathered. The conidia germinate and grow and develop into vegetative hyphae after they invade the silkworm. The vegetative hyphae are filamentous, 2.3 to 3.6 microns wide, with a septum, and can produce branches. During the growth of vegetative hyphae, short oval hyphae (cylindrical spores) can be formed on the top or side of the hyphae. The short hyphae are 5.6 to 16.8 × 2.8 to 3.08 microns in length and width, floating in the blood. Soon, one or both ends of the short hyphae elongated into vegetative hyphae, and at the same time, new short hyphae can be formed on the new hyphae, and they will spread to all tissues of the body along with the blood circulation (Figure 1). After the silkworm dies, the hyphae grow vigorously in various tissues, among which adipose tissue, Martens tube, silk gland, neurosphere and muscle tissue are more vigorous, but less invading and growing in the digestive tract. After 1 to 2 days, the vegetative hyphae in the body penetrate out of the body to form aerial hyphae. Aerial hyphae also have a septum and can branch and grow. Many conidiophores grow solitary or clustered on aerial hyphae, most of which bifurcate at right angles symmetrically with aerial hyphae. The conidiophore is bottle-shaped, with a large base and a small end. The tip is curved in a zigzag shape, and each bend extends into a very short small stalk. On the small stalk, there are conidia, which are grape-shaped after maturity. The bunches accumulate on aerial hyphae (Figure 1).

Beauveria bassiana can secrete toxins during growth. This toxin is a cyclic polypeptide compound. Several species have been found. Among them, Beauverin II is more toxic to silkworms, and it is a cyclic tetrapeptide. The artificial feed of 4th instar silkworms containing 4-8ppm Beauverin II can kill the silkworms. These toxins have the effect of complexing with cations, which can cause significant changes in the anion concentration of silkworm tissues, which is one of the reasons for the rapid death of silkworms. In addition to the production of toxins, octahedral calcium oxalate crystals can also be microscopically detected in the blood of diseased silkworms. Among the various sclerosis bacteria, Beauveria bassiana has the strongest pathogenicity to silkworms, and the disease is also the most acute.

Figure 1. Beauveria morphology

1. Conidia
2. Germination of conidia
3. Short hyphae (cylindrical spores, blastospores)
4. Conidia birth status

The conidia of Beauveria bassiana can survive for about 5 months to 1 year in places where there is no sunlight outside or in the soil; it can survive for 3 years at a low temperature below 10℃; it can survive for 4 years at a low temperature of -20℃ Long. Under direct sunlight (32～33℃), 5 hours can make it lose its pathogenicity. The resistance to disinfectants is much weaker than that of Aspergillus, immersing in 1-3% formaldehyde solution for 10 minutes, immersing in 0.2% effective chlorine bleaching powder solution for 5 minutes, immersing in 0.1% mercury solution for 2 minutes, and immersing in 70% alcohol It can be inactivated in 1 minute.

The main route of transmission of Beauveria is through body wall contact, followed by trauma. The conidia covering the carcass of beauveria bassiana are easy to fall off, and are light in weight and scattered with the wind. After the spores are brought into the silkworm seat and attached to the body wall of the silkworm, under suitable temperature and humidity conditions, they will begin to germinate in about 6-8 hours, the spores will expand, and then 1-2 germination tubes will grow. Germination tube can secrete proteolytic enzyme, lipolytic enzyme and chitinase. The body wall of the parasite is decomposed by the combined action of the three enzymes, and then the mechanical pressure of the extension and growth of the germination tube penetrates the body wall and invades the body of the parasite (Figure 2).

The optimum temperature for the germination and development of Beauveria bassiana conidia is 24-28°C, and neither can germinate nor develop under 5°C or above 33°C. At a suitable temperature, the germination of Beauveria bassiana must be above 75% relative humidity. The higher the humidity, the higher the germination rate. In the production of sericulture, the temperature and humidity in the silkworm seat is very suitable for the germination and development of Beauveria bassiana.

The conidia of Beauveria bassiana are eaten down, because the conidia cannot germinate in the digestive juice of the silkworm, and are excreted with the feces, and will not cause the silkworm to cause disease.

In the early stage of infection with beauveria bassiana, silkworms look the same as healthy silkworms. On the day before death, many oil-soaked or dark brown spots appeared on the body’s surface. The lesions appear in irregular locations and irregular shapes. The appearance of such lesions is caused by the degeneration of the chitin skin caused by the invasion of bacteria. When the amount of infectious bacteria is low, the appearance of lesions will be reduced or even no lesions will appear at all (Figure 3). Soon the sick silkworms lost their appetite rapidly, and some were accompanied by diarrhea and vomiting, and the silkworms were on the verge of death. At this time, if the blood is taken and examined under a microscope, many spores can be seen and the blood is slightly cloudy. After being infected with Beauveria bassiana, silkworms die in about 3 to 7 days. Small silkworms die faster, while large silkworms die slower; silkworms that start to infect faster and slower in the mid-feeding period; when pathogenic bacteria invade more quickly, Slow when less. If the infection is at the 5th instar, it will usually die at the end of 5th instar or even in the cocoon or in the cocoon.

Symptoms of Beauveria bassiana

The carcass of diseased silkworms gradually harden due to the development of parasitic bacteria. After 1 to 2 days, hyphae and conidia will grow. If they are left naturally for a certain period of time, many needle-like crystals will be precipitated from the body.

In the diagnosis of Beauveria bassiana, apart from the common characteristics of the dead silkworm, because the beauveria bassiana has oily spots, short oval hyphae can be seen in the blood under the microscope, and the corpse after death does not appear light red or form. The mycelium can be distinguished from other dead silkworms based on these characteristics, and a preliminary diagnosis can be made. Further, the diseased silkworm can be disinfected on the body surface (1% effective chlorine bleaching powder solution for 1 minute) to isolate the pathogenic bacteria, and finally identify whether it belongs to Beauveria bassiana.

The control of beauveria bassiana is mainly done by disinfection of silkworms before silkworm rearing and silkworm pedestals during silkworm stage. The purpose of disinfection of silkworm silkworm tools is to kill the conidia of pathogenic bacteria remaining on silkworm silkworm tools. Using formaldehyde disinfectant, Euchlorine disinfectant, bleaching powder disinfectant and ancient sulfur fumigation method, etc., can effectively kill the spores of Beauveria bassiana. The purpose of disinfection of the silkworm body and silkworm seat is to kill the conidia of Beauveria bassiana that has been brought into the silkworm seat or even attached to the silkworm body. Before 1970, organic amalgam anti-rigor powders such as Sailisan and Xilisheng were widely used. Later, the test and promotion of Sanlidan, Captan, Chlorothalonil, Fangbing No. 1, Fanggangling No. 2 and Excellent New anti-rigid agents such as Chlorine to replace organic mercury agents. These drugs, as long as they are used strictly according to the method of use, have good effects on the prevention and treatment of this disease. When using anti-rigid agents, it is generally sufficient to use it once before the ants and silkworms and their instars feed, but when this disease occurs, it is best to use it once a day, and the amount of powder to be seen on the silkworm seat is like frost. After dusting the powder, add a net to the mulberry after about 10 minutes. The fumigation method is also a simple and easy method to prevent stiffness, such as the application of pine needle fumigation. In the silkworm stage, hay, chaff and sawdust and other materials with high fiber content are often used to smoke. In addition, in order to prevent the occurrence of Beauveria bassiana, attention should be paid to repelling pests in the mulberry garden, timely sorting out diseased silkworms and frequently using dry materials, and prohibiting the use of Beauveria bassiana as a biological control agent in the silkworm area.