Introduction

Black Pepper is affected by several diseases caused by Fungi, Bacteria, Virus and Mycoplasma, besides nutritional disorder. Crop losses due to diseases and pests are identified as major causes of low productivity of pepper in India (Sarma and Anandraj 1995).  The  earlier record of diseases of pepper in India was that of Barber (1903, 1905).  Butler (1906) also  recorded the  death of pepper and Rao (1929) isolated phytophthora from diseased pepper Phytophthora capsici occurs on all parts of the plant and cause severe economic damage.  The symptoms expression depends upon the site of infection and extend of damage (Mammooty 1978, Anandraj and Sarma, 1995).  Aerial infection occurs on the summer shoots, foliage, spikes and branches causing blight, spike shedding, defoliation  and die back and at times death of the plants.  Infection on the runner shoots often reach the collar causing foot rot.  There is no effective control medicine to combat this malady.However, IISR Shakthi ( Released from IISR, Calicut ) is reported to be resistant to phythophthora capsici  while black pepper cultivars panniyur-1 and kaluvally reported to be susceptible  & tolerant  respectively to the disease  ( Kuch & Khew , 1982 and Sharma Nambiar ,1982 )  Hence, the present study was  aimed at analyzing anatomical basis of disease  resistant  among susceptible,tolerant and resistant genotypes of black pepper to phytophthora capsici.

Materials and Methods

The plant specimens for the proposed study (IISR Shakthi, Panniyur -1, Kalluvally) were collected from  IISR  Experimental Farm, peruvanamuzhi, Calicut, Kerala. Three varieties of P. nigrum VIZ, Panniyur-1 (susceptible) Kalluvally ( Tolerant ) I I SR Shakthi (Resistant) were selected for the study.  Rooted cuttings grown in polybags under green house  condition  (4 month old) were used.  Pure culture of P. Capsici isolated from infected black pepper leaves were  inoculated on carrot  agar medium in petriplates and incubated at 27⁰ C for  optimum  growth. Seven day old cultures having profuse growth were used for inoculation . Culture discs  of uniform size (5mm dia) were applied on the ventral side of the leaf lamina, stem, petiole after making pin  pricks (10 no:s) at the points of contacts.  The Mycellium was crushed with the mortar and pestle and was inoculated into soil. The Resistant, susceptible,Infected plant materials  were  cut and fixed  in FAA.(Formalin  5 ml + Acetic acid 5ml + 70% Ethyl Alcohol – 90ml).After 24 hrs of fixing,  the specimens were dehydrated with graded series of tertiary -Butyl  alcohol as per the schedule given by Sass, (1940).  Infiltration  of   specimens was carried by gradual addition of paraffin wax (melting point (58 – 60⁰) until TBA solution attained super saturation.  The specimens were cut into paraffin Blocks, which were sectioned  with the half of Rotary Microtome.  The sections were stained with Toluidine blue as per the method published by O’ Brien et al (1964).  Microscopic descriptions of tissues are supplemented  with Micrographs.  Photographs  of different Magnification were taken with NIKON  LABPHOTO2 Microscopic units. Magnifications of the figures are indicated by scale bars.  Descriptive terms of the anatomical features are given in the standard Anatomy Books (Esau, 1964).

Figure 1   Click here to View figure

Table 1: Comparison Of Anatomical Features Of Resistant and Tolerant Variety Resistant Variety   Tolerant  Variety

Structural Changes of the Infected Organs of Pepper

Leaf

The infected leaf exhibits thick continuous layers.  Cleistocarps, surrounded all around by thick dark covering.  The cleistocarps are in different stages of development, they are on the adaxial surface  of the leaf. In  some of the first bodies, there is an apical pole through which conidia are likely to be released.  This conidiophores  are thicker and dense.  The conidia are elliptical and elongated.  The subepidermal  cells break from the epidemis  and become crumbled.  The mesophyll tissues are also distorted due to the formation  of the first bodies on  the epidemis.

Petiole

The infection occurs in the adaxial groove of the petiole.  The lateral borders of the groove are wounded.  The cells along the borders undergo wound healing  process by producing this zone of small suberised cells.  This infection seems to penetrate the cells along the concavity so that some of the cells are crushed and others become dark and compressed.  Since the infected is in initial stage, interior tissue and vascular strands are not altered significantly.

Stem

In a young stem, the critical zone consists of only parenchymatous tissue and no sclerenchyma cylinder is seem.  So the inoculated pathogen has spread into wide corticial tissue, which become dark and crushed.  This stimulates poliferation and hyperplasia of the inner cortical cells adjacent to vascular bundles.

In the old stem, where there is a cortical cylinder of sclerenchyma  cells, the entry of the pathogen occurs by breaking sclerenchyma cylinder and later infection spreads to the outer  cortical tissues.   The fungal pathogen seems to be intra cellular.  The fungal organism is found occluding the cell cavity of the cortex.

During advanced stage of infection, the critical paramchyma cells on the pith, in the medullary rays, cortex and within  the vascular bundles get collapsed into dark amorphous  structures.  However, there is no evidence of presence of fungal pathogen  to the xylem elements of sclerenchyma cells.

Roots

In the root entry of the pathogen  is through an injury made in the cortical region.  It  spreads in the cortical cells and then enters into the stele.  More and more cortical cells become invaded by the pathogen and the cells are turned into dark structure.  The most obvious effects of the pathogen is manifested in the xylem elements.  The xylem elements are occluded by dark, amorphous substances which evidently blocks the flow of water  to the aerial system of the plant.

Conclusion

The comparative account of the anatomical features of resistant variety  and susceptible  variety reveals certain structural differences between these two. Previous  studies by Marks and Mitchell (1971), Miller and Maxwell (1984), Philips etal (1987), Hamalavi etal (1995), and Markose (1996), on various host plant infested by phytophthora have established that thicker epldermis, smaller no: of epidermal appendages, smaller cortical cells, smaller stele, thicker pericyclic cells,  vascular bundles and compact arrangement of cells can be attributed to phytophthora infection.

In the present study we have obtained results similar to those mentioned above in the resistant variety of piper  nigrum as compared to susceptible variety . physical barriers found in the resistant variety seams to play a pivotal role in resisting fungal infestation. That it can be concluded that anatomical differences observed in 3 piper nigrum genotypes can be used for screening various genotypes against  tolerance / resistant to phytophthora capsici  The selection of tolerant / immune genotype via screening will be helpful to evolve resistant / tolerant plant and also on the selection  of plants for further breeding.

Acknowlwdgement

This paper forms a part of PhD thesis ( Plant biology & Biotechnology submitted by Miss Remmia Raghavan to University of Madras,  Nov 2010. Deeply acknowledged to the Director IISR experimental form Peruvanamuzhi Calicut, Kerala for providing the plant materials for study.    

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