The pattern of pathogen diversity and abundance in Lentil ( Lens culinaris ) fields in Constantine region , Algeria

Lenses are a group of pulses having a socio-economic and nutritional significance. The study was conducted on two fields of Lens culinaris in Constantine region contaminated with molds, had the objective to put a relationship between pathogenic molds associated with these plants and their environment. This study revealed the presence of 20 genera in soils (Absidia, Acremonium, Alternaria, Aspergillus, Bysochlamyces, Chaetomium, Cladosporium, Emericella, Eurotium, Fusarium, Mucor, Paecilomyces, Penicillium, Peronospora, Phytophotora, Pseudalesheria, Scopulariopsis, Scytaldium, Trichoderma and Ulocladium) and 20 genera also in plants (Absidia, Acremonium, Alternaria, Aspergillus, Botrytis, Chaetomium, Cladosporium, Cylindrosporium, Curvularia, Eurotium, Fusarium, Myrothecium, Onychocola, Phytophthora, Pseudalescheria, Penicillium, Peronospora, Rhizoctonia, Trichoderma and Ulocladium). They contribute approximately 54% of the total micropopulation enumerated in studied samples. The development of these pathogenic strains is governed by environmental conditions namely the chemical elements in soil, pH, electrical conductivity, Nitrogen, Carbon and saturation. The results we have obtained shows that the chemical variations ground contribute to the right development fungi and their transfer to plants


INTRODUCTION
Lentil is a dicotyledonous legume plant and takes the fifth place in production of pulse crop in the world (Hymowitz, 1990).Lentil is still widely used today because of its high fiber, protein, vitamin and mineral content (Hnatowich, 2000) and considered as one of the best vegetable source of Iron especially important for adolescent and pregnant women.Furthermore, lentil is cultivated in sandy loam soil and can be grown in nutrient deficient soil (Summerfield, 1981).Lentils are drought resistant and can be grown in water logged and saline soils (Muehlbaur et al., 2002).
In Constantine, lentil production has tripled in the last six years from 37,120 kg in 2006 to 710,074 kg in 2013.A survey of literature showed that many fungal species have been reported for lentil seeds including species of Pythiumsp, Rhizoctoniasp, Sclerotiumsp, Fusariumsp, (Muehlbaur et al., 2002), Alternariasp, Aspergillussp, Mucorsp, Chaetomiumsp, Penicillium sp.Nigrosporasp (Hussain et al., 2007), Uromycesfabae and Botrytis sp.(Richardson, 1979).However, the presence of so much pathogenic fungi reduces the quality and quantity of crop which in turn lead to economic losses.
Soil is the most important resource of the nature (Sumithra et al., 2013), it is a thin layer of the earth's crust which serves as a natural medium for the growth of plants because the components of soils are mineral material, organic matter, water and air.Soil has important ecological functions in recycling resources needed for plant growth and microorganisms' development, the soil forms a large reservoir for many potential plant pathogens and especially plants with a decreased vitality (Katarzyna and Christel, 2011).An extremely simplified vegetation, such as a monoculture, selects a specific microbial community, including plant pathogenic microorganisms and sometimes also their parasites or antagonists (Bruggen et al., 2006).
This study aimed to reveal the complex interactions within the infected soil-lentil ecosystem and the aggressive behavior of molds.

Studied area
The field experiments were conducted in 2011 and 2012 on two agricultural soils: AIN SEMARA and BARAOUIA situated in the North East of Algeria.

Soil sampling
Soil samples taken at 5, 10 and 15 cm from the soil surface were collected from four locations in the vicinity of the two areas (AIN SEMARA and BARAOUIA), in sterile paper bags.The soils collected are ground and passed through 0.2 mm sieve and were used for the analysis.

Plant sampling
Lentil plants from Metropole variety (20 samples), were collected from different places in fields infected by many types of mold.These plants were collected in sterile paper bags.

Physical and chemical properties analysis
The physiochemical properties are executed in a laboratory of soil chemistry.The important parameters studied are: available Nitrogen, Organic Carbon, Electrical conductivity and pH value.

Organic Carbon
The Walkey and Black (1934) method described by Nelson and Sommers in 1982 is used for assaying the organic carbon in simple soils.

Electrical conductivity
The assay method involves wet ground with 100 g of distilled water slurry.The paste obtained was centrifuged at 300 revolutions / min for 30 min.In the end, the temperature of the float is noted in the resistance, is measured after adjusting the temperature to 25°C, using a conductivity meter (Anonymous, 1954).

pH
The method relies on the preparation of a solution (diluted suspension) of ground 10 g and 25 mL of distilled water, after magnetic stirring, the pH is measured at room temperature using a pH meter (Boudoudou et al., 2009).

From soil (Dilution plating method)
To isolate the fungi from the soil sample, 1 g was diluted in 9 mL of saline solution.1 mL of the diluted sample was poured and spread on Petri plates containing sterilized PDA medium.The inoculated plates were incubated at a temperature of 27°C for 3 days (Warcup, 1950).To prevent bacterial growth, one milligram of chloramphenicol was added to the medium.

From plants
Infected plants collected from the websites surveyed (AIN SEMARA and ABARAOUIA), was cut into small pieces of about 0.5 cm.The pieces are disinfected with sodium hypochlorite (2%) for 3 min, then rinsed several times with sterile distilled water and ethanol (70%) and dried between sterile filter paper, then placed in Petri dishes (5 piece / Petri plates) containing a culture medium PDA, MEA5 or MS.Finally, the Petri plates were incubated at 27°C for 4 days (Belabid et al., 2000).

Presentation of data
The number of genera is referred to as genera diversity.Population density is expressed in terms of colony forming unit (CFU) per gram of soil with dilution factors.In order to assert the dominance of the genus in site, percentage contribution was worked out as follows: Total number of all colonies of all the genera in a sample

Physical and chemical properties analysis
The results of the physical and chemical properties analysis of two soil samples (with different depth), have revealed that soils of AIN SEMARA and BARAQUIA are rich in organic matter (>1) and by consequence Carbon and Nitrogen.In addition, the organic matter is rich in carbon and poor in Nitrogen.Furthermore, results of pH analysis show that values are slightly alkaline (between 7.69 and 7.86).The measure of electrical conductivity (EC) and the salinity factor are indicated to lower than 1; which means that the soils were not salty (Table 1).

From soil
Altogether, six soil samples from two different stations representing the entire Constantine district were examined for fungal diversity.The study resulted in the presence of 44 species of fungi belonging to 14 genera from BARAOUIA and 42 species of fungi belonging to 16 genera from AIN SEMARA (Figure 1).Indeed, the soil sample of BARAOUIA is characterized by the presence of 68.21% Deutromycetes, 20.43% Ascomycetes, 6.82% Oomycetes and 2.27% of Phycomycetes and Zygomycetes.The soil samples of AIN SAMARA contain less Deutromycetes (50%), Ascomycetes (35.72%) and Zygomycetes (9.52%) but more Oomycetes (4.76%) (Figure 2).

DISCUSSION
The samples of soil analysis were taken from the first horizon (0 to 20 cm) in which the of the biological activity is concentrated.This horizon is the most exposed one to the air and it contains mainly aerobic species.The internal environmental conditions can assign the specific composition of the microbial communities and their chemical potential (Alexander, 1997).Additionally, with an organic-matter equal to 1.79% in (BARAOUIA) and 1.81% in (AIN SEMARA), the studied soils are included in the interval of normal rates (1.5 to 2.5%) which are described in the standards of Duthil (1970).
The comparison of the pH values of the soil studied with the data standards by Madagascar (quoted by the interprocessor of the agronomist (1974)), shows that our soils are slightly alkaline.According to Baise and Jabiol (1995), the alkali soils have a pH between 7.3 and 8.5.These pH values should be perfect for fungi development.
The values of the electrical conductivity (EC) measures of the soil which are taken from BARAOUIA and AIN SEMARA show that the soils are not saline (EC< 1) and this is according to the norms given by DURAND (quoted by the interprocessor of the agronomist, 1974).Furthermore, the C/N report that was calculated after the determinations of Carbon and Nitrogen, has given the results <15, which means that the organic material has been normally mineralized.Thus, it provided mineral nitrogen.
The analysis of the results obtained after the isolation of the fungi of the two prospected sites shows that 84.09% of the strains isolated from the BARAOUIA site (genera: Acremonium, Alternaria, Aspergillus, Chaetomium, Cladosporium, Fusarium, Perenospora, phytophtora, Penicillium, and Ulocladium) and 90.73% of the AIN SEMARA site (genera: Acremonium, Alternaria, Aspergillus, Eurotium suggests, Penicillium, Phytophthora, Fusarium, Pseudallescheria, Ulocladium and Trichoderma) are isolated from the different parts of infected plants and also from the sampled soils.These results showed that the strains have been transmitted from the soil through the sap which can take for granted the soil as a middle of storage of fungal spores.These results are in relationship with those of Estelle Levetin and also of Abigail Jenkis (2005).
Furthermore, the remains of the soil isolates (15.91% of BARAOUIA and 9.27% of AIN SEMARA) are plants only.It means that these strains are transmitted through the seed or the air (Estelle Levetin).
Actually, the study of the different isolates shows that only 55.34% of the strains of the BARAOUIA site (genera: Alternaria, Botrytis, Cladosporium, Cylindrosporium, Fusarium, Peronospora, Phytophthora and Rhizoctonia) and 46.3% of the strains of the AIN SEMARA site (genera: Alternaria, Cylindrosporium, Phytophtora and Fusarium) are of pathogenic strains associated with Lens culinaris plants.These results are in coincidence to those of Richardson (1979), Ahmed et al. (1993), Muhlabauer et al. (2002) and Hussain et (2007).
The great diversity of isolates obtained after identifications (soil and plants) is mainly due to the environmental conditions favouring the development of molds: rate of rainfall between 19 and 68 mm, 60 to 71 humidity and temperature between 19 and 26 o C. The majority of the identified isolates belong to large families of the Ascomycetes and Deuteromycetes.These strains are indigenous, usually isolated from the ground (Alvarez-Rodriguez et al., 2002).

Figure 1 .Figure 2 .
Figure 1.Examples of some species isolation from soils and plants.

Figure 3 .Figure 4 .Figure 5 .
Figure 3. Percentage of contribution of different genera in the soil of BARAOUIA and the soil of AIN SEMARA.

Table 1 .
Results of physico-chemical analysis of soils in Constantine region.