PACKAGE OF PRACTICES FOR MULBERRY CULTIVATION AS TREE PLANTATION FOR RAINFED SERICULTURE

PK. Das, SB. Magadum and DS. Chandrasekhar
Dr.P.K.Das is Scientist-D at Regional Sericultural Research Station (RSRS), Central Sericultural Research & Training Institute, Central Silk Board (CSB), Government of India Chamarajanagar, Karnataka, India. He has put up a service in Central Silk Board, for more than 28 years. He worked for more than 20 years at Central Sericultural Research & Training Institute (CSR&TI) Mysore in Agronomy laboratory and developed a number of technologies for cost effective mulberry cultivation. Among his interests are recycling technologies of sericultural wastes, compost and vermicompost, Azotobacter and VA-mycorrhiza as biofertilizers and developing Integrated Nutrient Management for mulberry cultivation. He was selected as the best scientist of moriculture of CSR&TI, Mysore in the year 2005. At RSRS Chamarajanagar he is developing technologies for rainfed mulberry. He has more than 100 research publications in national and international journals and guided four Ph.D students in Applied Botany under the university of Mysore. He has also visited Japan under Japan International Co-operation Agency (JICA) project of Central Silk Board during the year 2000. Dr. Das can be contacted at pkd3@rediffmail.com

Dr. S.B. Magadum is Scientist-D with CSR&TI, Mysore, India. He has more than 35 year’s research experience. A veteran silkworm physiologist, he has published more than 140 research publications in national and international journals. Dr. Magadum can be contacted at sbmagadum@rediffmail.com  

Dr. D.S.Chandrashekar is Scientist –D and in charge of RSRS, Chamrajanagar. He is an expert of mulberry pathology. He has more than 33 year’s research experience. He is currently supervising 6 Ph.D students and have more than 100 research publications in national and international journals. Dr. Chandrasekhar can be contacted at rsrschnagar@rediffmail.com  

What is tree mulberry?

The mulberry plants which are allowed to grow tall with a crown height of 5 - 6 feet from the ground level having stem girth of 4 -5 inches or more is called tree mulberry. They are specially raised with the help of well grown saplings of 8 - 10 months old with any of the varieties recommended for rain fed areas like S-13 (for red loamy soil) or S-34 (black cotton soil) which are tolerant to draught or soil moisture stress conditions. Usually the plantation is raised as block plantation with a spacing of 6 feet x 6 feet or 8 feet x 8 feet as plant to plant and row to row distance. The plants are usually pruned once in a year during monsoon (July - August) at a height of 5 - 6 feet from the ground level and allowed to grow with maximum of 8 - 10 shoots at crown. The leaf is harvested 3-4 times in a year by leaf picking method under rain fed or semi-arid conditions depending upon the monsoon.

Fig-1: Two years old mulberry tree plantation

Concept of tree plantation:

Combining trees and field crops in arable lands is called as "agro-forestry". The objective of agro-forestry is to improve the productivity and sustainability of land management system through introduction of woody perennials in herbaceous crop husbandry. Selection of tree species to be used in agro forestry must be based on cultural and economic as well as environmental and biological factors. Thus growing mulberry as tree in highly eroded flat to gentle sloppy land unfit for growing arable crops, arable lands with soil fertility problems, degraded sloppy land can serve as one of the best means of agro-forestry.

Who can grow tree mulberry?

Mulberry is a perennial plant with reasonably drought tolerance capacity. It can be cultivated as a trained tree by maintaining specific spacing between trees and crown height. This type of mulberry plantation is highly suitable for farmers of semi-arid / rain fed areas in plain land, hilly regions or in denuded lands unsuitable for agriculture. This form of cultivation is already been practiced in temperate and hilly regions. Of late, the concept of tree cultivation has also spread into the plains as a sustainable crop under severe water stress condition in waste and denuded lands. With the recommended packages, it is now possible to get about 6 - 8 MT of mulberry leaf yield per hectare per year through 3 - 4 harvests. It is also possible to take up appropriate inter crops in the tree mulberry plantation to reap higher economic benefits. Considering the above, mulberry can be cultivated under rain fed conditions with different systems of cultivation. These include;

(a) Bush system of mulberry cultivation: under protective irrigation.

(b) Low-height tree type mulberry cultivation: Suitable in hilly regions.

(c) Tree mulberry cultivation: Best suited to overcome acute water stress conditions.

Package of practices for mulberry tree plantation:

Development of mulberry tree saplings:

The mulberry saplings are developed in the nursery. A flat land nearer to water source is preferred as nursery site. Well drained land with loamy soil is ideal for nursery. The land must be ploughed or dug 30‑40 cm deep and allowed for weathering in sun for 2 - 3 weeks. Land is again ploughed two or three times to bring the soil to fine tilth. Root stocks, pebbles and weeds are removed at the time of ploughing and the land is leveled. The land is divided into a number of small beds to prepare the nursery. The size of each bed is decided keeping working convenience in to mind. A bed size of 3.0 m (L) x 1.2 m (B) accommodates 100 cuttings (row to row 30 cm and cutting to cutting in a row 10 cm distance) to raise 8-10 months old saplings. Each bed on all sides is separated by a bund of 25 to 30 cm width and height and provided with irrigation channel of 25 to 30 cm width and 15 to 20 cm depth. Each bed should be manured and mixed thoroughly with 5 pans of FYM / sericulture compost / vermicompost. In the case of clayey or black cotton soil, additional 5 pans of sand per bed should be mixed with soil uniformly. In the case of red loamy or sandy loam soils, there is a possibility of termite infestation. As a preventive measure, 0.1 % Chloropyriphos (5 ml per litre of water) can be sprayed to drench the soil of nursery beds (2-3 litres per bed). Regular care and irrigation should be provided for good growth.                

 Fig-2: Mulberry nursery under preparation

Transplantation of saplings from the nursery

For tree plantation, the saplings are transplanted from the nursery after 8-10 months of maturation. The matured saplings are removed from the nursery by deep digging and without damaging the roots. It is advisable to irrigate the nursery beds thoroughly at least 2-3 days before uprooting to facilitate easy and complete removal of saplings with roots intact. The uprooted saplings are immediately planted in the main field after removal of leaf, top clipping and dipping the roots of the plants in 0.2 % solution of Diethane -M 45 to avoid fungal root disease.

Fig-3: Appropriate mulberry cuttings for nursery to grow as saplings

Plantation of saplings in the main field & establishment care:

A flat / sloppy land with red loamy / black cotton soil or denuded land not suitable for other agricultural crops can be selected for raising tree mulberry as block plantation. Plantation can be taken only during rainy season preferably in July - September or depending upon the onset of monsoon. The land should be thoroughly ploughed by tractor / bullock plough depending upon the soil condition after receiving one or two pre monsoon shower and weeds should be removed.

Fig- 4: One year old mulberry saplings ready for transplantation as tree plantation.

Once the land is made ready, farmyard manure / sericulture compost can be applied @ 10 MT/ ha and mixed with the soil. It is highly necessary to follow soil moisture conservation practice by raising wide bunds all along the four boundaries of the plantation to avoid runoff and allow rain water percolation in the planted area during monsoon. Before plantation, pits of the size of 35 cm (L) x 35 cm (B) x 35 cm (D) are dug at 8 feet apart from each other considering plant to plant and row to row distance as 8 feet x 8 feet. Each pit is then planted with one transplanted matured sapling exactly in the centre of the pit. To determine the centre of the pits and to keep the rows straight to avoid zigzag plantation, two ropes are used length and breadth wise and the intersecting point of the two ropes is considered as the centre of each pit. The pits are then filled with soil and pressed properly for better anchorage with the ground. Once the plantation is over, all the planted saplings are pruned uniformly at 5 feet height (crown height) from the ground level within 2-3 weeks and allowed to grow for 8-10 months as establishment period or even a year without harvesting leaf / disturbing the plants. However, weeding should be done as and when required during the establishment period to facilitate better growth. After 4 -5 months of plantation, the first weeding is done manually or by using power tiller to avoid damage and chemical nitrogen fertilizer only @ 50 kg per hectare is applied to boost the growth of plants. The required fertilizer in the form of urea / ammonium sulphate is applied near each plant by making basin and irrigation is followed. If required gap filling can be made with properly grown sapling. Plants should be given life saving irrigation as and when required in non rainy period for better establishment. Further, the whole planted area can be divided in to small blocks of 15 - 20 plants in each having wide bunds all along the four sides to allow in-situ soil moisture conservation during rainy season. During the establishment period, the plants may attain a height of 10 -15 feet from the ground level with three to four branches if properly maintained.

Fig-5: Mulberry tree plantation under progress in main field.

Pruning and package to be followed from the second year:

Once the plantation is established properly, the plants are pruned uniformly at the same crown height (5 feet) where the plants were pruned earlier during the time of plantation. This should invariably be followed only during rainy season (July - September) to facilitate the vigorous growth of shoots from the second year onwards. Farmyard manure @ 10 MT per hectare per year is applied within a week of pruning and the weeding is followed with the help of tractor / power tiller / country plough to mix the manure with soil and to save the manual lobour days. Immediately after this basins around the plants are cleaned to apply fertilizers and allowing rain water percolation near the plants. Depending upon rainfall, chemical fertilizer NPK is applied @ 150:60:60 kg per hectare per year in two equal splits in the form of ammonium sulphate for alkaline soils or urea for acidic soils in early and later part of rainy season. Green manuring with sunhemp or dhaincha for the improvement of soil fertility and water holding capacity or intercropping with short duration crops (Groundnut, Cowpea, Horsegram , Ragi etc) for augmenting income can also be done from the second year onwards. If the plantation is inoculated with VA-mycorrhiza followed by green manuring, reduced dose of FYM & NPK fertilizer can be applied @ 10 MT and 50:25:25 kg per hectare per year respectively. Leaf can be harvested by individual leaf picking after every three months depending upon the rainfall and soil moisture condition. Thus it is possible to harvest 3 - 4 crops annually ranging from 7 - 8 MT of leaf per hectare per year.

Fig-6: Pruned mulberry saplings after one year of plantation.

ASPERGILLOSIS IN SILKWORM

S. Manochaya and J. Justin Kumar
S. Manochaya is a budding researcher, currently working as a project fellow in Biodervisity Bioprospecting and Sustainable Development under Institute of Excellence in Department of Microbiology, University of Mysore. A post graduate in Microbiology, she had earlier worked with M/s. Healtline Pvt. Ltd., Sericare Division, Bangalore as R &D Officer and involved in DBT project “Silk Protein blend film for burn wound management” and in the development of other neutraceutical products. She is interested in Agricultural and Medical Microbiology for herbal drug development. Mr. J. Justin Kumar works with Central Sericultural Research and Training Institute, Mysore, India. His research interests include silkworm pathology and application of bio-molecules in silkworm disease management. They can be contacted at: mcs_nks@yahoo.co.in & justinkumarj@gmail.com

Introduction

In tropical countries like India the success of silkworm rearing depends upon the protection of crop from the disease causing pathogens. In temperate sericulture regions ideal climate, superior quality of mulberry leaves and restricted number of rearing in a year reduces the chance of disease incidence. The reason for outbreak of silkworm diseases in India includes continuous rearing throughout the year, availability of large population of different stages in a limited area, inferior quality of mulberry leaves and unhygienic rearing condition etc. Disease out break not only affects the income of the farmers but also scuttles the seed cocoon and silk production plans. Domestication for the past several thousand years has rendered the silkworm, highly susceptible to different pathogens leading to diseases and crop loss. Different micro-organisms such as viruses, bacteria, fungi and microsporidia cause infectious diseases in silkworm.

Aspergillosis

Aspergillosis is a major silkworm disease in several sericultural countries. This is caused by a number of Aspergillus species of fungi. Aspergillus species have been known to be pathogenic to silkworms since the latest part of 19th century (Nomura, 1897). This disease is commonly called `Kojickabi’ in Japan (Ayuzawa, et al., 1972) and also known as brown muscardine. Many aspergillus species have been reported to infect silkworm (Aoki, 1971). More than 10 species of Aspergillus were reported from Thailand, Indonesia, Srilanka and India (Govindan and Devaiah, 1995) as pathogenic to silkworm, viz., A. flavus, A. tamari, A. oryzae, A. niger, A. ochraceus, A. sojae, A. fumigatus, A. nidulans, A. flavipes, A. clavatus, A. terreus, A. melleus, A. elegans, A. parasiticus, etc. A. flavus Link and A. tamari Kita are most common in India. Aspergillus bombycis Peterson et al. is a recently described species known only from domesticated silkworm [Bombyx mori L. (Lepidoptera: Bombycidae)] culture in Indonesia and Japan (Peterson et al. 2001).
Aspergillus is a facultative fungus and is able to live saprophytically in the silkworm rearing environment like soil surface and rearing appliances, silkworm faeces etc (Aoki, 1971; Ayuzawa et al., 1972). These form the source of the fungus and thus disease spreads rapidly. Though Aspergilli are saprophytic, they are reported to be pathogenic to several insects in addition to Bombyx mori (Govindan et al., 1998). The early instars i.e., first and second instar silkworm larvae are more susceptible and later stage silkworms are fairly resistant to this diseases. High temperature and high relative humidity conditions maintained during young stage are reportedly contributing factors to greater disease incidence during young age (Govindan and Devaiah, 1995). Aspergillus flavus Link. and Aspergillus tamarii Kita are commonly found strains in India.

Systematic position of the pathogen 

The Genus Aspergillus belongs to the Family Trichocomaceae, Order Eurotiales, Class Eurotimycetes, Phylum Ascomycota, Kingdom fungi and Domain Eukarya. Though there are hundreds of species of Aspergillus, all are not pathogenic to silkworm. The systematic position of the genus is:

Domain: Eukarya

Kingdom: Fungi

Phylum: Ascomycota

Class: Eurotiomycetes

Order: Eurotiales

Family: Trichocomaceae

Genus: Aspergillus

Mode of infection

Majority of aspergilli grow well and sporulate abundantly at 23 - 26°C (Thom and Raper, 1945). Temperature between 30 - 35°C for Aspergillus flavus and 20°C for A. tamarii are congenial for good growth (May et al., 1931). The thermal death point is 54°C for A. flavus and 55°C for A. tamarii. According to Kawakami (1982), temperature range of 28 - 30°C and relative humidity from 85 to 95% favours the growth of silkworm, which in turn is congenial for aspergillus development. 100% mortality occurs at all combinations of temperature (20, 25 and 30°C) with humidity levels (80, 85, 90 and 95%) (Chinnaswamy, 1983). The disease development is slow at low temperature and more rapid at high temperature.

Aspergillus spp. infects the silkworm through the integument. The conidia are the infectious units and these on coming in contact with host-integument under congenial conditions of temperature and humidity germinate to put forward the germ tube that penetrates through integument.

The penetration is generally observed in the inter-segmental region, the top of leg, the connecting part of seatae and around spiracles. In the low pathogenic fungi, the penetration is significantly delayed or unable to penetrate the larval integument. The germ tube after entering through the epithelium branches at the spot of entry. At the point of entry black marking may be noticed. The fungus does not form short hyphae as in case of B. bassiana and grows only at the site of infection and finally the larva die due to the secretion of aflatoxin. At the hardened site, aerial hyphae protrude to form the conidiophores. The conidiophores are thick and expand into a globular or oval structure called apical vesicle, bearing 1-2 rows of radiating sterigmata. The conidia attach in chains to the sterigmata. The conidia are light and are easily dispersed to spread the disease

Symptoms

Since Aspergillosis is caused by various species of Aspergillus, the symptoms are not always same. Young silkworms are very much susceptible, compared to the mature worms. Infected larvae stop feeding, become letharginc, show body tension and lustrousness and the victim die soon due to Aflotixin produced by the fungus in the host. Aerial hyphae appear a day after death and later conidia cover the body giving particular colour according to the Aspergillus species. Hardening of the body is limited to the site of infection and the rest of the body decay. Diagnosis of the disease is based on the hardening of the corpse and the morphology of the hyphae.

Silkworms infected with A. flavus

                        

             Silkworm infected with A. tamarii

Physiological changes in infected larvae

The pathogen has been observed to produce kojic acid (C6H6O4). The virulence of the strain of A. flavus and their resistance to formalin is related to the production of Kojic acid (Kawakami and Mikuni, 1969). Two toxic fractions also have been isolated from A. flavus that kills the larvae of mosquitoes Culex peus and C. tarsalis (Toscano and Reeves, 1973). The Aspergillus species also produce toxins called aflatoxins. The aflotoxins are very potent carcinogens and produce tumours primarily in the liver of vertebrates, including human being (Wyllie and Morehouse, 1977). Four aflotoxins B1, B2, G1 and G2 have been detected from A. flavus. Aflotixin B1 was highly toxic and G1 was moderately toxic to silkworm larvae. There is correlation between the presence of aflotoxin and the pathogenicity to the silkworm, their tolerance to formalin, and the ability to produce pigments (Ohtomo et al., 1975). A strain of A. flavus in addition to aflotoxin, also produce two more toxins, cyclopiazoic acid (indole tetramic acid) and aflatrem (indole mevalonate meatbolite) (Richard and Gallagher, 1979). A. ochraceus which also infects silkworm produces destruxins (Kodaira, 1961). Toxins produced by A. fumigatus include fumagillin, helvolic acid, fumitremorgins, phthioic acid and gliotoxin among others [http://www.aspergillus.man.ac.uk/index home.htm (accessed on 11/06/2010)].

Incidence and loss
Chinnaswami (1983) reported that in Karnataka, India the percentage of disease incidence ranged from 5.32 (February-March) to 21.36% (July-August). In Thailand, incidence of Aspergillosis is more during June and August and less during September (Aoki, 1971). The disease is noticed on first to third instar larvae during January to February and on fourth and fifth instar larvae during July (Aoki et al., 1972). Singh et al., (2004) reported that the maximum crop loss per 100 layings (50000 larvae) was 1.35±0.73 to 1.61±1.46 kg cocoons. Incidence was higher in bivoltine silkworm rearing than in multivoltine and cross breed.

Disease management

Preventive measures such as disinfection and hygiene maintenance in the rearing environment is the best way to keep the disease at bay. Many studies have proved that many A. flavus strains are resistant to formaldehyde (CH2O). Hence the use of disinfectant should be judiciously chosen. Sodium penta chloro phenoxide monohydrate as a disinfectant against Aspergillus fungi invaded into rearing tools was more effective than 3% formalin (Wadee et al., 1972). Benzalkonium chloride, iodine disinfectant, benzalkonium chloride + dodecyl diaminoethylglycine, didecyl dimethyl ammonium chloride, etc are proved to be effective (Kawakami, 1982). In vitro studies by Graham and Graham (2007) have shown that, mycelial growth and toxin production by A. parasiticus were inhibited by garlic concentration of 0.3 - 0.4%.  Sun drying of rearing equipments is an effective way of destroying Aspergillus pathogens to some extend. Sick worms discovered before conidification should be incinerated or placed in lime jars and never thrown around indiscriminately. The faeces and bed refuses should be disposed off properly and disinfection with anti muscardine powder should be carried out immediately.

Research gaps

Aspergillosis is not considered to be a very serious threat to the sericulture industry. But the sparseness of studies on the extent of its damage and economc loss has lead to gross under estimation of its ill effects. This research gap deserves attention. Development of silkworm breeds tolerant to aspergillosis could be a worthwhile pursuit for silkworm breeders. Further the persistence and crossing over of Aspergillus pathogens from other insect pests and mulberry pests needs to be thoroughly investigated.

References
Aoki K (1971) Silkworm diseases in Thailand. Bull. Thai. Seric. Res. Trg. Inst. 1: 102-108.
Aoki K, Isarangkul L and Sinchaisri N (1972) On silkworm diseases, especially pebrine and Aspergillus diseases found in 1971. Bull.Thai Ser.Res.Trani. Centre, 2: 72-76.

Ayuzawa C, Sekido T, Yanakawa K, Sakura V, Kuratta W, Yaginuma Y and Tokora Y (1972). Agricultural Techniques manual-1. Handbook of Silkworm rearing, Fuzi publishing Co. Ltd, Tokyo, Japan.

Chinnaswamy, KP (1983) Studies on Aspergillosis of silkworm Bombyx mori Linnaeus caused by A. tamari kita M.Sc. (Agri) Thesis, UAS Bangalore, p. 112.

Govindan R and Devaiah MC (1995) Aspergillosis of silkworm. Silkworm Pathology Technical Bull No.1. Dept. of Seri. UAS Bangalore, p:68
Graham HD and Graham EJF (2007) Inhibition of Aspergillosis parasiticus growth and toxin production by garlic. J. Food Safety 8(2): 101-108.

Govindan R, Narayanaswamy TK and Devaiah MC (1998) Principles of silkworm pathology, Seri Scientific publishers, Bangalore, pp. 270-285.

Kawakami K (1982) Causal Pathogens of Aspergillus diseases of silkworm and its control, JARQ, 15 (3): 185-190.

Kawakami K and Mikuni T (1969) Studies on the causative fungi of Aspergillus disease of the silkworm larvae I. Pathogenicity to the silkworm larvae and tolerance to formalin of Aspergilosis isolates collected from co-operative rearing houses of young silkworm larvae. Bull. Seric. Expt. Stn. (Tokyo) 23: 327-370.

Kodaira Y (1961) Toxic substances to insects, produced by Aspergillus ochraceus and Oospora destructor. Agric. Biol. Chem. (Tokyo), 25: 261-262.

May OE, Moyer AY, Wells PA and Iterrick MT (1931) The production of Kojic acid by Aspergillus flavus . J. American Chem. Soc .53: 774-782.

Ohtomo T, Murakoshi S, Sugiyama J and Kurata H (1975) Detection of aflotoxin B1 in silkworm larvae attacked by an Aspergillus flavus isolate from a sericultural farm. Appl. Microbiol. 30: 1034-1035.

Richard JL and Gallagher R (1979) Multiple toxin production by an isolate of Aspergillus flavus. Mycopathologia, 67: 161-163.
Singh GP, Selvakumar T, Sharma SD, Nataraju B and Datta RK (2004). Estimation of crop loss due to aspergillosis and pathogenicity of Aspergillus to Silkworm, Bombyx mori L. Sericologia 44(3): 321-326.

Thom C and Raper KB (1945) A manual of the Aspergilli. The Williams and Wilkins Co.; Baltimore, M.D; USA. p. 373.

Toscano NC and Reeves EL (1973) Effect of Aspergillus flavus mycotoxin in Culex mosquito larvae. J. Invertebr. Pathol. 22: 55-59.

Wadee L, Ayuthaya I, Sinchaisri N and Aoki K (1972) Disinfection of Aspergillus grown on silkworm rearing tools made of bamboo. Bull. Thai. Seri. Res. Trg. Centre, 2: 72-76.

Wyllie TD and Morehouse LG (1977) “Mycotoxic Fungi, Mycotoxins, Mycotoxicoses: An Encyclopedic Handbook”. Vol. 1, Marcel Dekker, New York.

SILK ROAD- HIGH WAY THAT LINKED CULTURES

SILK is one of the most mysterious creations of Mother Nature. Its history is as often said, “Shrouded in mystery and legend.” Both Indian and Chinese versions of history exist. There are references of the fabric in ancient scriptures of both the countries. However it is generally believed that silk found its origins in China, more than four thousand years ago. The Chinese legend says that it was the teen aged Chinese empress “Hsi-Ling-Chi” of the yellow emperor “Huang Te” who brought the secrets of silk to light. Historic relics unearthed from China support this legend. The Chinese still worship the empress as the “Silk Goddess”.

For many centuries Chinese kept the art of silk making a close guarded secret and tantalised the whole world with the ‘heavenly fabric’. ‘Anyone who tried to smuggle either silkworm eggs or mulberry seeds was bound to meet death penalty’. No wonder even learned Roman like Pliny the Elder fancied silk as “the hair of sea-sheep”. Emperor Justinian of Byzantium (Istanbul) employed two monks to smuggle out silkworms from China in 550 AD. They accomplished the task by hiding live specimens inside hollow canes. By 1000BC the Chinese silk products became so popular that they started export, mainly through caravans on foot and camel back. Later this caravan tract came to be known as SILK ROAD.

Through Silk Road, silk travelled from China to almost every part of the world. In return wools, gold and silver went to China. It is to be born in mind that this happened in a time when most parts of the globe remained un touched by man and those touched lay un connected. Today man has acquired the Godly attribute of ‘omnipresence’ by means of internet and other gadgets. A look back to 3000 BC reveals the fact that it is not the gadgets that work wonders but human imagination, courage and will power. Let us see how the Silk Road progressed, what did it do for man and what is its present state.

After the demise of Huang Te, the Yellow emperor, China was ruled ‘Han Dynasty’. It was during Han Dynasty, silk industry reached its Zenith in China and Silk Road originated. The road started from Shanghai on the Pacific Ocean and traversed along the Great wall through ‘Sian’. Sian, then a cosmopolitan Chinese city had a population of two million and excelled as a trade centre. At the mouth of the ‘Taklimakan desert’, the road split into two, each branch binding the sides of the desert. Collin Thubron, who re- traced the silk road in 1989 says “the desert is one of the most dangerous voids on earth. You enter and never return”. The two branches re united at Kashagar- the last great oasis within China. Even now Kashagar is a hotspot of trade between Pakistan and China. It is interesting to note that the trade route even linked Kashmir, then a great trading centre. From Kashagar the road rose into the Russian Pamir, again splitting into two. One branch lead to Samarkand and the other to Afghanistan. Once again they reunited near the frontiers of Iran. After travelling through Baghdad, Damascus and Istanbul, the road broke on the shores of Adriatic ocean. The goods were then shipped across to Rome. Subway followed from Rome to Genoa and Cadiz in Spain, where the road ended. From Shanghai to Cadiz the Silk Road measured nearly 12800 km. It remained the longest man-made road on earth during the 2000 years of its use.

Apart from its role in international trade, the Silk Road has tremendous historic significance. It is the first connecting link between the west and the east. It facilitated spread and amalgamation of various cultures, caused rise and fall of empires and created new countries and civilisations. The most important contribution however was to the growth and spread of various religions. Indian pilgrims travelling Silk Road introduced Buddhism to China. It also helped spread of Judaism, Islam and Christianity across Asia. To quote the Encyclopaedia Britannica, “literary records do not reveal much about the process but the comparatively abundant information surrounding the birth of Islam in Arabia [AD 610-32] casts much light on the sort of religious exchanges that might have occurred in caravan camps and round innumerable campfires where strangers met telling tales and expounding divergent beliefs”.

With the gradual decline of Roman Empire in Asia and the rise of Arabian power, the Silk Road became increasingly unsafe and untraveled. With the opening of a sea route between Europe and India the importance of the land route declined. In the 13th and 14th centuries t was revived under Mongols and Marco polo used the road to travel to China. The road now partially exists in the form of a paved highway, connecting Pakistan and China. The old road has inspired a United Nations plan for a trans-Asian highway. An oil pipeline 3017 km. Long is constructed along the Silk Road from China to Kazakhstan.

The humble silkworm too has travelled much. From the Chinese queen’s tender bosom the creature has been smuggled out, transported across great mountains and seas and across a variety of climates, cultures and civilizations. Now China has lost its monopoly, for no knowledge could be kept secret for long. Knowledge is nobody’s property. It is for the good of the whole mankind.

Analysis of amino acid content of semi-synthetic diet (seri-nutrid) and mulberry leaf and their requirement for growth of silkworm, Bombyx mori L

Kanika Trivedy, Sunderamurthy Nirmal Kumar and Manchala Ramesh

Dr. Kanika Trivedy is a scientist with the Central Silk Board, India. She has rendered more that 25 years of service as a researcher and teacher in Sericulture sciences. Her major work is in the field of silkworm endocrinology and in developing artificial diet suitable to Indian conditions. In consideration of her outstanding contributions in this field she has been awarded the WIPO Gold medal and certificate for “Best woman Inventor of the year 2004” by World Intellectual Property Organization, Geneva , “Technology day award 2004” by Government of India and award on “Annual day celebration 2008” from Director, CSR&TI, Mysore, for the outstanding contribution in the field of sericulture research. Dr. Kanika Trivedy has published a large number of papers in international and national reputed journals. An avid silkworm enthusiast and a gifted teacher of silkworm biology, Dr. kanika is a warm and pleasant personality. She can be contacted at: kanikatrivedy@gmail.com

INTRODUCTION

Amino acids as precursors of proteins are essential to all organisms including the silkworm. Literature on nutritional significance of amino acids in silkworm Bombyx mori reveals the quantitative and qualitative requirement of the amino acids (Arai and Ito, 1964, Ito and Arai, 1967, Bose et al. 1989 and Nair and Kumar, 2004). Amino acids which bind together as long chains in proteins will be broken down to amino acids in the process of digestion mediated by the digestive enzymes in the midgut. The amino acids thus separated are released into the bloodstream and will be utilized by individual cells to assemble new and different proteins required for specific functions. Silkworms do not have enzymes required to synthesize all the amino acids. The six amino acids which silkworm can produce are proline, alanine, glycine, serine, tyrosine and cystine. Essential amino acids required for synthesis of silk are obtained only through food and they are Arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine (group 1) and Aspartic acid and glutamic acid (group 2). These amino acids are required for the growth of the larva.

The present investigation was undertaken to know the available amino acids in the formulated and cooked diet and also to compare with mulberry leaf amino acid content. Further, comparative bio assay studies were taken up to determine the quality of diet so that suitable modifications if any can be made to improve the Seri-nutrid on the basis of its amino-acid content.

MATERIALS AND METHODS: The semi synthetic diet for silkworm was formulated at CSRTI, Mysore and licensed to M/S SERICARE, Bangalore for commercial production. The product which is being sold in the trade name "SERINUTRID" was used for the present study for the analysis of amino acid content and compared with mulberry leaf amino acid content.

Sample preparation: 100g Seri-nutrid (Wet diet) dried and dry mulberry leaf (V1 variety) was hydrolyzed with 6 N hydrochloric acid at 40°C for 36 hours. The hydrolyzed sample was centrifuged and the supernatant was collected for 3-4 times by washing with water. Further HCl was removed and treated with activated charcoal to remove the colour of the sample. The sample with activated charcoal was kept overnight and then filtered to get colourless sample and analyzed for amino acid content.

Analysis: Free amino acids and protein-hydrolyzed amino acids were analyzed by cation exchange column Shim-pack ISC-07/S1504 (4.0mm id x 15 cm) and the amino acids were detected by post column derivation with o-phthalaldehyde (OPA) and spectrofluorophotometric at 450 nm using Shimadzu HPLC system. Sample solution was derivatized. This cation exchange method permits analysis of wide variety of amino acids ranging from free amino acids to amino acids produced in hydrolysis of protein. Each amino acid was separately run as standard for its chromatographic identity and in a mixture to reconfirm its time of elution and area for its known concentration. The response factors were calculated from multiple injections of a known standard run concurrently with the samples.

Laboratory evaluation:Commercial Multivoltine x Bivoltine hybrid PM x CSR2 was used for bioassay studies. For diet rearing, room temperature was maintained at 30°C where as for leaf rearing it was 28°C. Three replications of 5 dfls (appx 2500 larvae) each of PM x CSR2 hybrid were brushed on diet following the procedure developed by Trivedy et al., (2003) and on leaf following the procedure of Rajan & Himantharaj (2005). Daily weight gain of larvae (10 worms weight) was recorded. The larvae were reared on Seri-nutrid and leaf separately up to II moult. After II moult, the larvae were resumed to III instar with V1 mulberry leaf and the rearing was continued till spinning with V1 leaf. Logistic growth curves, larval growth rate and growth indices were calculated.

Large-scale field-testing: Three trials of 800 dfls each were conducted with commercial PM x CSR2 hybrid at farmers place through selected Chawki Rearing Centres using Seri-nutrid (produced by M/S SERICARE, Bangalore). Simultaneously leaf control of the same quantity in each trial was maintained. The diet and leaf reared young instar worms were distributed to 19 and 18 farmers respectively. Further rearing continued at farmers place with V1 leaf till spinning. Yield data was collected and analyzed statistically.

Statistical analysis: Linear regression (R2), Student t test for growth parameters and Logistic regression for growth curves.

RESULTS AND DISCUSSION

The present study was undertaken to evaluate the performance of Seri-nutrid with respect to its amino acid content and compared with the performance of conventional leaf rearing. The amino acids are white crystalline solids at normal temperature; when heated to high temperature, they decompose rather than melting. They are stable in aqueous solution and with few exceptions can be heated up to 120°C for short periods without decomposition even in acid or alkaline solution. Thus the hydrolysis of proteins was carried out under the said conditions with the complete recovery of most of the constituent and free amino acids. The results revealed that the hydrolyzed sample of Seri-nutrid contains arginine (arg), histidine (his), lysine (lys), phenylalanine(phe), methionine(meth), threonine (threo), leucine (leu), isoleucine(isoleu) and valine(val) to the tune of 9.07, 1.75, 4.55, 3.71, 0.22, 2.68, 6.79, 3.00 and 4.15 g% where as in leaf, it is to the tune of 1.28, 0.44, 1.40, 1.28, 0.29, 1.18, 2.05, 1.05 and 1.30 g% respectively (Table 1a & 1b) except tryptophan present in traces could not be identified. All the essential amino acids of Seri-nutrid are more in quantity as compared to leaf except methionine which is almost equivalent to that of leaf. Similarly, the quantity of II group of essential amino acids viz. Aspartic acid (7.88) and Glutamine (18.22) in Seri-nutrid are more compared to leaf i.e. 2.88 and 2.55 g % respectively. Proline is considered as semi-essential amino acid which is also more in Seri-nutrid. Similarly, other non-essential group 1 and 2 amino acids are more in Seri-nutrid as compared to leaf (Table 1a and 1b). Minimum requirement of essential amino acids of group I viz., arg, his, leu, isoleu, lys, meth, phe, threo, trypto and val are to the tune of 46, 32, 61, 61, 55, 27, 48, 59, 10 and 69 µg, group II essential amino acids Aspartic acid 40-65µg and Glutamic acid 65µg and non-essential group II amino acids Tyrosine 60-110µg and Cystine 110 µg/g dry diet respectively for Silkworm (Source: Technical reports of Sericultural Experimentation Station, Japan). Silkworm can produce 7 of the 19 amino acids. Other amino acids must be supplied through the food. In case, if the silkworm fails to obtain enough quantity of any one of the twelve essential amino acids, it will result in degradation of the body proteins and muscle. Unlike fat and starch, the silkworm body do not store excess amino acids for later use; the amino acids must be supplied through the food every day. Free amino acids are found in the body fluids of silkworm in amounts that vary in different tissues. The amino acids glutamic acid, aspartic acid and their amides which play key role in the incorporation and transfer of ammonia are often present in relatively high amounts whereas the concentration of other amino acids of proteins are extremely low ranging from a fraction of a milligram to several milligrams per 100 g wet weight of tissue. The requirement of ten common amino acids (arg, his, isoleu, leu, lys, meth, phe, threo, trypto and valine) and proline in silkworm has been demonstrated by the deletion of single amino acid from a mixture (Arai and Ito, 1964, Ito and Arai, 1965 & 1967). Proline was considered to be essential for Bombyx mori, without which neither normal growth nor development was observed but its requirement was less than the first ten essential amino acids. Thus proline was considered as semi-essential amino acid (Arai and Ito, 1967). Inokuchi et al. 1967 composed diet with amino acid mixture and nutritive effect was tested by 11 amino acids singly. When one of the 10 essential amino acids was omitted, there was no growth and development. High mortality was observed in diet lacking with Arg, Meth or Valine. They confirmed that the proline is very important for growth and development. After the improvement in composition of semi-synthetic diet, silkworms were raised entirely on amino acid diet (Ito and Arai, 1965) and reported the requirement of amino acids in silkworm and proved the importance of aspartic and glutamic acid. Inokuchi (1970) studied the quantitative effects of single omission of dietary amino acid on the protein level and ninhydrin positive substances in the haemolymph of the silkworm larvae using synthetic diet. Further the author explained the changes in the level of free amino acids in haemolymph of the larvae fed on diet lacking one amino acid and the relation between the levels of free amino acid of haemolymph. Inokuchi(1970) further studied the amino acid content of the leaf and diet and compared with haemolymph amino acid content of leaf and diet fed silkworms. It was found that depending upon the quality of food, quantity of each haemolymph amino acid changed (Ito et al. 1967).

Logistic growth curves were plotted for both the treatments (Figures above) and R² values (goodness of fit) were calculated by linear regression method. R² values indicated that there is no significant difference between the treatments (0.976 for Serinutrid and 0.975 for V1 leaf) which indicated a perfect fit between the treatments. Further, the similarity in logistic curves between the treatments gave a clear indication about the quality of semi-synthetic diet “Seri-nutrid” and can be safely used for young instar rearing. Larval growth rate and growth indices were also calculated and presented in Table 2a & 2b. The results indicated non significant difference in growth rate of I instar larvae between Seri- nutrid and V1 mulberry where as in II instar, V1 reared larvae showed significantly higher growth indicating the scope for improving the Seri-nutrid for II instar worms. This can be achieved by improving the amino acid transport system in larvae by adding few enzymes as studied by Leonarda et al. 2001. According to Leonarda et al. (2001), leucine methyl ester (Leu-OMe) can increase the activity of transport system responsible for the absorption of most essential amino acids of the diet in the larval midgut which significantly influences larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight, 12-18 h before the control larvae and produced cocoon shells up to 20% heavier than those of control. Activation of amino acid absorption plays vital role in larval development. Amino acids can adjust or control the substance metabolism and physiological function of silkworm by combining the active substance like enzyme, hormone etc, with other substances.

Growth indices revealed that, initial growth in Seri-nutrid reared batches was lower than V1 leaf reared batches, but non-significant difference between the treatments was noticed from III instar onwards. Larval duration and cocoon traits were same in both the treatments except shell %. Seri-nutrid reared batch recorded low shell percentage (Table 2c) because of its high pupal weight.

Large-scale bioassay data revealed non significant difference in cocoon yield, cocoon weight, shell weight and shell % between the treatments (Table 3). The reeling data (Table 4) indicated that the diet-reared batches are equally good in all the reeling parameters tested.

Analysis of amino acids and their ratio in mulberry leaf protein and cocoon silk protein revealed non significant difference. Requirement of either glutamate or aspartate and non-essential amino acids for full growth, and partially replacement of dietary alanine, glycine and serine by glutamate and aspartate was studied by Ito and Arai, 1966 & 1967. Their study indicated that growth of silkworm greatly depends not only on the level of amino acids in the diet but also on the mutual balance of amino acids. Horie et al., 1970 studied the effect of balance of three amino acid groups viz. essential, non-essential and acid amino acids on larval growth and cocoon quality and concluded that tryptophan and phenylalanine could be synthesized by their precursors. By the use of artificial diets, nutritional requirement of silkworm has been elucidated.

Thus Seri-nutrid, a semi-synthetic diet can be safely used for young instar rearing up to II instar. Though it contains high amino acid in hydrolyzed sample, amino acids available after digestion of protein of Seri-nutrid in midgut are sufficient for normal growth of I instar silkworm and it proved that availability of amino acid in diet is at par with the requirement for the robust growth of silkworm and to the amino acid content of the mulberry leaf in first instar. Whereas, improvement in the diet composition for II instar larvae can be achieved by adding few enzymes which can activate the amino acid absorption in the midgut thereby improving larval weight gain in second instar. Based on the these results it can be concluded that there is no need to add extra amino acids as Seri-nutrid itself contains sufficient quantity of amino acids required for silkworm growth and development.

Table 1a: Amino acid contents in Seri-nutrid (g/100g dry weight).

Essential gr. 1		Essential gr. 2
Arginine	9.07	Aspartic acid	7.88
Histidine	1.75	Glutamic acid	18.22
Lysine	4.55	Semi- essential
Tryptophan	-	Proline	9.81
Phenylalanine	3.71	Non essential gr.1
Methionine	0.22	Alanine	6.38
Threonine	2.68	Glycine	14.92
Leucine	6.79	Serine	4.76
Isoleucine	3.00	Non essential gr. 2
Valine	4.15	Tyrosine	2.27
		Cystine	0.009

Table 1b: Amino acid contents in V1 mulberry leaf (g/100g dry weight).

Essential gr. 1		Essential gr. 2
Arginine	1.28	Aspartic acid	2.88
Histidine	0.44	Glutamic acid	2.55
Lysine	1.40	Semi- essential
Tryptophan	-	Proline	1.12
Phenylalanine	1.28	Non essential gr.1
Methionine	0.29	Alanine	1.15
Threonine	1.18	Glycine	1.36
Leucine	2.05	Serine	1.24
Isoleucine	1.05	Non essential gr. 2
Valine	1.30	Tyrosine	0.79
		Cystine	-

Table 2a: Lab trial data (a) Average growth rate (g / day) value of silkworm, Bombyx mori, PM x CSR2.

Instar	Seri- Nutrid	V1 mulberry leaf	‘t’ Value
I Instar	0.0163	0.0180	8.167
II Instar	0. 1326	0. 1433	51.401

Table Value @ 5% for 4 degree of freedom is 2.776

Table 2b: Lab trial data (b) Growth Index value of silkworm, Bombyx mori, PM x CSR2.

Treatment	I Instar	II Instar	III Instar	IV Instar	V Instar
Seri-nutrid	14	70	378	1732	10778
V1 mulberry leaf	14	75	398	1733	10779
‘t’ value	ns	3.062	12.247	1.225	0.144

Table Value @ 5% for 4 degree of freedom is 2.776

Table 2C: Lab trial data (c) Larval duration and cocoon traits value of silkworm, Bombyx mori, PM x CSR2.

Treatment	Larval Duration (Days:Hours)					Cocoon traits
	I Instar	I Moult	II Instar	II Moult	Total Larval Duration	Cocoon weight (g)	Shell weight (g)	Sell percentage (%)
Seri-nutrid	3.00	1.02	2.00	1. 06	24.05	1.751	0.322	18.38
V1 mul leaf	3.00	1.02	2.00	1.06	24.05	1.725	0.320	18.52
‘t’ value	ns	ns	ns	ns	ns	7.723	1.225	8.573

ns - Non-Significant

Table Value @ 5% for 4 degree of freedom is 2.776

Table 3: Rearing performance of silkworm, Bombyx mori, PM x CSR2 in field

Treatment	Average Yield Kg/100dfls	Rate (Rs)	Cocoon weight (g)	Shell weight (g)	Sell percentage (%)
Seri-nutrid
Mean	68.07	123.81	1.743	0.327	18.76
SD±	2.81	9.46	0.16	0.03	0.27
Leaf
Mean	67.24	124.87	1.718	0.326	18.99
SD±	3.24	3.35	0.05	0.01	0.85
t - test	0.337	0.182	0.254	0.065	0.452

Table Value @ 5% for 4 degree of freedom is 2.776

Table 4: Reeling performance of the cocoons of hybrid silkworm, Bombyx mori,

PM x CSR2.

Parameters	Diet reared batches	Leaf reared batches	t - test
Filament length (m) (FL) Average Non-breakable Filament length(m) (NbFL) Average Denier Reelability % Rendita A (kg) Rendita B (kg) Raw Silk Recovery % Waste % on silk weight	790±10 692±4 2.69±0.21 88.17±1.2 7.4±0.21 7.8±0.13 66.18±0.10 17.6±0.21	814±14 720±6 2.78±0.18 88.9± 1.1 7.1±0.18 7.3±0.20 68.3±0.89 19.4±0.20	2.42 6.73 0.564 0.777 1.88 3.63 4.10 10.8

Table Value @ 5% for 4 degree of freedom is 2.776

REFERENCES

Arai, N. and Ito, T., 1964, Amino acids requirements of silkworm Bombyx mori L. J. Seric. Sci. Jpn., 33 (2): 107-110.

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Bose P. C., Majumder S. K. and Sengupta., K., 1989, Role of amino acids in silkworm,Bombyx mori L. Nutrition and their occurrence in haemolymph, silk gland and silk cocoons-a review. Indian J. Seric., 28 (1): 17-31.

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Ito T. and Arai, N., 1967, Nutritive effects of alanine, cystine, glycine, serine and tyrosine on the silkworm Bombyx mori. J. Insect Physiol., 13 (12): 1813-1824.

Ito, T., Arai N. and Inokuchi, T., 1967, Nutrition of silkworm Bombyx mori XVII Effects of dietary levels of amino acids on growth of fifth instar larvae and on cocoon quality. Bull. Sericul. Exp. Sta., 21 (2): 398-400.

Leonarda M. G., Casartelli, M., Fiandra., L., Parenti P. and Giordana, B., 2001, Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition. Arch. Insect Biochem. Physiol., 48: 190-198.

Nair J. S. and Kumar, S. N., 2004, Artificial diet for silkworm (Bombyx mori L) – A retrospection through the decades. Indian J. Seric., 43(1): 1-17.

Rajan. R. K. and M. T. Himantharaj, 2005. Silkworm rearing technology. Sampath J (ed), Central silk board, published by Basker, H. pp 61-110.

Trivedy, K., Nair, K. S., Ramesh, M., Gopal N. and Kumar, S. N., 2003, New Semi-synthetic diet “Nutrid” – A technology for rearing young instar silkworm in India. Indian J. Seric., 42 (2): 158-161.

AKNOWLEDGEMENTS: Authors are thankful to the staff of silkworm Physiology for assisting in experimental silkworm rearing and owner of CRC for conducting the large-scale diet young instar rearing.