Enzymes are the large biomolecules that are required for the numerous chemical interconversions that sustain life. They accelerate all the metabolic processes in the body and carry out a specific task. Enzymes are highly efficient, which can increase reaction rates by 100 million to 10 billion times faster than any normal chemical reaction. Due to development in recombinant technology and protein engineering, enzymes have evolved as an important molecule that has been widely used in different industrial and therapeutical purposes. Microbial enzymes are currently acquiring much attention with rapid development of enzyme technology. Microbial enzymes are preferred due to their economic feasibility, high yields, consistency, ease of product modification and optimization, regular supply due to absence of seasonal fluctuations, rapid growth of microbes on inexpensive media, stability, and greater catalytic activity. Microbial enzymes play a major role in the diagnosis, treatment, biochemical investigation, and monitoring of various dreaded diseases. Amylase and lipase are two very important enzymes that have been vastly studied and have great importance in different industries and therapeutic industry. In this review, an approach has been made to highlight the importance of different enzymes with special emphasis on amylase and lipase in the different industrial and medical fields.

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A Broader View: Microbial Enzymes and Their Relevance in Industries, Medicine, and Beyond

Domestication, Genomics and the Future for Banana

Banana (Musa spp.) is one of the world's most important fruits. In 2011, 145 million metric tons, worth an estimated $44 billion, were produced in over 130 countries. Fusarium wilt (also known as Panama disease) is one of the most destructive diseases of this crop. It devastated the 'Gros Michel'-based export trades before the mid-1900s, and threatens the Cavendish cultivars that were used to replace it; in total, the latter cultivars are now responsible for approximately 45% of all production. An overview of the disease and its causal agent, Fusarium oxysporum f. sp. cubense, is presented below. Despite a substantial positive literature on biological, chemical, or cultural measures, management is largely restricted to excluding F. oxysporum f. sp. cubense from noninfested areas and using resistant cultivars where the pathogen has established. Resistance to Fusarium wilt is poor in several breeding targets, including important dessert and cooking cultivars. Better resistance to this and other diseases is needed. The history and impact of Fusarium wilt is summarized with an emphasis on tropical race 4 (TR4), a 'Cavendish'-killing variant of the pathogen that has spread dramatically in the Eastern Hemisphere.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-04-15-0101-RVW

Fusarium Wilt of Banana

Lipases (triacylglycerolacyl hydrolases, EC3.1.1.3) are class of enzymes which catalyze the hydrolysis of long-chain triglycerides. In this review paper, an overview regarding the fungal lipase production, purification, and application is discussed. The review describes various industrial applications of lipase in pulp and paper, food, detergent, and textile industries. Some important lipase-producing fungal genera include Aspergillus, Penicillium, Rhizopus, Candida, etc. Current fermentation process techniques such as batch, fed-batch, and continuous mode of lipase production in submerged and solid-state fermentations are discussed in details. The purification of lipase by hydrophobic interaction chromatography is also discussed. The development of mathematical models applied to lipase production is discussed with special emphasis on lipase engineering.

Overview of fungal lipase: a review.

The advancements made in tissue culture techniques has made it possible to regenerate various horticultural species in vitro as micropropagation protocols for commercial scale multiplication are available for a wide range of crops. Clonal propagation and preservation of elite genotypes, selected for their superior characteristics, require high degree of genetic uniformity amongst the regenerated plants. However, plant tissue culture may generate genetic variability, i.e., somaclonal variations as a result of gene mutation or changes in epigenetic marks. The occurrence of subtle somaclonal variation is a drawback for both in vitro cloning as well as germplasm preservation. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage. Several strategies have been followed to ascertain the genetic fidelity of the in vitro raised progenies comprising morpho-physiological, biochemical, cytological and DNA-based molecular markers approaches. Somaclonal variation can pose a serious problem in any micropropagation program, where it is highly desirable to produce true-to-type plant material. On the other hand, somaclonal variation has provided a new and alternative tool to the breeders for obtaining genetic variability relatively rapidly and without sophisticated technology in horticultural crops, which are either difficult to breed or have narrow genetic base. In the present paper, sources of variations induced during tissue culture cycle and strategies to ascertain and confirm genetic fidelity in a variety of in vitro raised plantlets and potential application of variants in horticultural crop improvement are reviewed.

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Somaclonal variations and their applications in horticultural crops improvement

In vitro propagation produces banana clones that arc very diverse. For 'Giant Cavendish', in addition to resistance to Fusarium wilt, the tissue culture method also generated clones with increased resistance to strong wind, heavier fruit bunches, and sweeter fruit. Therefore, it may be an ideal procedure for horticulturists to select banana clones producing fruit with different taste and might be possible for plant pathologists to select clones resistant to other important diseases. The possibility of applying this technique to the improvement of other crops remains to be exploited. Although 40,000 'Cavendish' plants grown from suckers did not show any visible difference in morphology, about 3 percent of 'Cavendish' plantlets derived from tissue culture were variants. Relatively little is known about the cause of genetic instability induced by the in vitro vegetative propagation. Rapid multiplication and development of cells resulting from mediation of regulators in the tissue culture medium may increase the chance of variation. The mechanism by which the somaclones of 'Giant Cavendish' are resistant to Fusarium wilt is unknown. Since the parental 'Giant Cavendish' is very susceptible to Fusarium wilt, the appearance of resistant somaclones may result from activation of silent resistant genes. However, the creation of resistance genes through mutation as the origin of the resistant phenotype cannot be ruled out. DNA technology will be useful in deciphering the true nature of wilt resistance in the future.

https://apsjournals.apsnet.org/doi/pdfplus/10.1094/PDIS.2004.88.6.580

Cavendish Banana Cultivars Resistant to Fusarium Wilt Acquired through Somaclonal Variation in Taiwan

In 1985, the third author (W. H. Ko) noticed unusually high mortality of some fruit trees in residential areas in southern Taiwan. Tree death appeared to be very sudden, because all the leaves had turned brown but had remained attached to the branches (Fig. 1). In 1987, numerous dying longan trees (Dimocarpus longan) were found in the mountain areas of central and southern Taiwan. Trees in the early stages of decline exhibited a brown discoloration of the wood on large roots (Fig. 2). A fastgrowing, basidiomycete-like fungus was subsequently isolated from diseased roots, and inoculation studies verified its pathogenicity. However, it was not possible to identify the pathogen at the time because fruiting bodies were not initially found on diseased trees in the field or in laboratory cultures, nor were clamp connections observed on hyphae. In 1990, Ko found a young, thin, flat, brown basidiocarp with a white margin on an exposed root of a diseased longan tree. He then realized that what looked like dirty bark on the basal stems of some declining trees was in fact remnants of decomposed fruiting bodies of the pathogen (Fig. 3) and that fungal structures previously thought to be mycelial mats on sawdust medium were actually fruiting bodies (Fig. 4). Subsequently, the fungus was identified as Phellinus noxius (Corner) G. H. Cunn. Soon after the report of brown root rot causing serious decline in longan (2), the P. noxius pathogen was found to be widespread and destructive on a number of tree species throughout Taiwan. Due to the seriousness of the problem, the Taiwan government created a Phellinus noxius Brown Root Rot Diagnosis and Control Information Center in 2000 to address public concern about the disease. History and Occurrence of the Disease Worldwide The fungus causing brown root disease of trees was first described in Singapore by Corner in 1932 as Fomes noxius (18) and reclassified by Cunningham in 1965 as Phellinus noxius (19). Corner speculated that the fungus was the cause of brown root rot of rubber trees and tea bushes. However, attempts to demonstrate pathogenicity of this fungus were not successful until the realization that it requires a food base for successful infection of the host. Bolland (11) inoculated hoop pine (Araucaria cunninghamii) by inserting a severed root into a jar containing 250 g of enriched sawdust medium colonized by P. noxius. The fungus killed the inoculated root and began to girdle the tree within 6 months. P. noxius has a wide host range; it has been reported on more than 200 plant species representing 59 families. Although most hosts are woody plants, some herbaceous plants are also susceptible to the pathogen. P. noxius is widespread among tropical countries in Southeast Asia, Africa, Oceania, Central America, and the Caribbean (25). It has also been reported from the tropical Hainan Island of China (29). In Japan, the pathogen was found only on the subtropical island of Okinawa (1). In Taiwan, it has been found in both tropical and subtropical districts (8,17).

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Phellinus noxius Brown Root Rot of Fruit and Ornamental Trees in Taiwan.

Media selective for the isolation of bacteria, actinomycetes and fungi were amended with 0.1% sunflower oil emulsified with 0.01% Tween 80. Lipase-producing microorganisms produced clear zones on these media. When lipase-producing bacteria were cultured on a polycarbonate membrane laid on the selective medium for bacteria, clear zones were produced on the medium when the membrane along with bacteria was removed. The agar disc cut from the clear zone also produced a clear zone when placed on the fresh medium, indicating that clear zone formation is the result of the activity of extracellular lipases. The largest population of lipase-producing microorganisms in an agricultural soil was actinomycetes followed by bacteria and fungi. Ranging from 12 to 75% of bacteria, actinomycetes and fungi isolates from soils collected from three different locations were capable of producing lipases. In general, relatively small percentages of soil bacteria were lipase producers, and lipase producers were more common among soil actinomycetes and fungi. These three groups of microorganisms appear to be all important in decomposition of oils in organic matters in soils.

A simple method for detection of lipolytic microorganisms in soils

Kumquat (Fortunella margarita (Lour.) Swingle) is an important citrus fruit crop of Iland County in northeastern Taiwan. Fruit produced in this area are mainly for making preserves, which is a well-known product in Taiwan. Decline of kumquat was first noticed in 1990. Since 1995, it has become an important problem affecting ≈80% of kumquat orchards. The problem was especially serious after the passage of a typhoon. Some orchards were abandoned due to death of many declining trees. Initial symptoms were yellowing and browning of leaves on some branches of affected trees. Abscission of leaves and fruits occurred, subsequently resulting in the appearance of dieback of affected branches. Disease symptoms could progress to other branches of the same tree, gummosis on the trunk, and eventually death of the tree. Previously, Phytophthora citrophthora (Smith & Smith) Leonian was reported to be a causal organism of kumquat decline (1). Recently, isolations from declining kumquat trees in several orchards failed to recover P. citrophthora and instead we isolated Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (syn. Botryodiplodia theobromae Pat.) from tissue taken from the margin of discolored bark and wood on symptomatic branches. The fungus produced grayish black colonies on V8 agar and black ostiolate pycnidia (125 to 650 μm in diameter) with ovoid to elongate conidia (20 to 32 × 12 to 16 μm) on autoclaved whole wheat grains that were placed on V8 agar. Young conidia were hyaline and nonseptate, whereas mature conidia were brown, one septate, and striate. Pathogenicity tests were carried out on healthy kumquat trees located at the Taiwan Agricultural Research Institute experimental farm. Three isolates of L. theobromae were cultured on wheat-oat medium. Colonized grains (≈5 g) were held against a wound made by lightly scrapping a branch (5 to 7 mm in diameter) 200 to 250 cm above the ground by wrapping a sheet of clear plastic around the branch (2). Eighteen branches were used for each isolate, and the same number of branches similarly inoculated with sterile grains as a control. Healthy branches inoculated with L. theobromae showed disease symptoms similar to those observed on naturally infected plants. Discoloration of leaves on inoculated branches occurred within 1 week. Subsequently, all the leaves fell and infected branches died. The numbers of inoculated branches killed by the three isolates of L. theobromae tested were 18, 9, and 14 after 1 month. All control branches remained disease free. L. theobromae was reisolated from symptomatic tissues, completing Koch's postulates. The same isolates were used to inoculate trunks of kumquat trees with the method described above. Six trunks were inoculated with each isolate, and the same number of trunks similarly inoculated with sterile grains was used as the control. Gummosis on inoculated trunks occurred in 1 week. The numbers of inoculated trunks showing gummosis induced by the three isolates of L. theobromae tested were 6, 5, and 6 after 1 month. L. theobromae was reisolated from symptomatic tissue. All control trunks remained free of gummosis. Our results show that in addition to P. citrophthora, L. theobromae can also cause a dieback on kumquat. To our knowledge, this is the first report of disease caused by L. theobromae on kumquat or on any species in the Rutaceae family in Taiwan. References: (1) P. J. Ann et al. Plant Pathol. Bull. (Taiwan) 6:198, 1997. (2) W. H. Ko et al. Plant Pathol. 35:254, 1986.

Wen-hsiung Ko

Papers

Cavendish Banana Cultivars Resistant to Fusarium Wilt Acquired through Somaclonal Variation in Taiwan

Phellinus noxius Brown Root Rot of Fruit and Ornamental Trees in Taiwan.

A simple method for detection of lipolytic microorganisms in soils

Lasiodiplodia theobromae as a Causal Agent of Kumquat Dieback in Taiwan