**1. Introduction**

106 Fungicides for Plant and Animal Diseases

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There is growing interest in the use of medicinal plants and herbal products for fungal infection related diseases control program because plant derived drugs are considered safe and free from adverse side effects. Plants generally produce many secondary metabolites which constitute an important source of antifungal drugs. Medicinal properties of plants are normally dependent on the presence of certain phytochemical principles such as alkaloids, anthraquinones, cardiac glycosides, saponins, tannins and polyphenols which are the bioactive bases responsible for the antimicrobial property (Ebana *et al*., 1993). It is difficult to discover antifungal agents against yeasts and filamentous fungi compared to bacteria caused diseases. Fungal cell wall is predominantly composed chitin and other polysaccharides such as β-glucans which become hindrance to the antibiotic activity on the cells. Hence appropriate screening methods to study the antifungal activity of natural resources play an important roles in the development of fungicide.

Plant drugs still remain the principal source of pharmaceutical agents used in orthodox medicine (Khaing, 2011). The ability of plant to produce various types of phytochemicals such as alkaloid, flavonoid and saponin attract the attention of natural products researcher. Although many plant or herbal products use for the treatment of various ailments in rural communities but there is a need for scientific verification of their activities against fungi. Currently, there is little scientific evidence on the antimicrobial properties of these medicinal plants under investigation against majority of fungi (Mann et al., 2008). *In vitro* and *in vivo* antifungal activities of medicinal plants have been studied widely in the present's days. A wide variety of methods can be applied to study antifungal activity. However, the outcome of these studies relies on appropriate and reliable methods use by the researchers.

In this chapter we analyze and compare the various reliable methods available for the study of antifungal activity. In this chapter, we describe and discuss plant sample extraction technique, antifungal screening with determination of minimum inhibition concentration, minimum fungicidal concentration and IC50 value on hyphal growth inhibition. Beside that the *in situ* antifungal study method by using various microscopy techniques such as confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and

Screening Methods in the Study of Fungicidal Property of Medicinal Plants 109

First steps in the process of screening medicinal plants for antifungal activity is extraction. Extraction is the separation of medicinally active portions of plant tissues using selective solvents through standard procedures. Such extraction techniques separate the soluble plant metabolites and leave behind the insoluble cellular marc. The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or in dry powder form, and are intended for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts or powdered extracts (Sukhdev et al., 2008). The basic operations of extraction include steps, such as pre-washing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Proper actions must be taken to assure that potential active constituents are not lost, distorted or destroyed

The general techniques (Fig. 2) of medicinal plant extraction include maceration (Fig. 3), infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet). Recently, modern extraction methods have been developed which includes microwave-assisted extraction (MAE), ultrasound extraction (sonication) and supercritical fluid extraction (SFE)

> **Coventional extraction methods**

**Modern extraction methods** 

**Soxhlet extraction**

**Sonification**

**Maceration**

**Supercritical Fluid extraction** 

**Microwave assisted extraction**

**Pressurized Liquid Extraction,**

during the preparation of the extract from plant samples.

Fig. 2. Conventional and modern extraction methods

**Extraction** 

**2. Extraction** 

(Table 1).

transmission electron microscopy (TEM) also will be discussed. Fig. 1 shows the various steps involved in the evaluation of the medicinal plant's for fungicidal properties.

Fig. 1. Various steps involved in the development and evaluation of fungicidal property of medicinal plants
