Fusaria and other fungi taxa associated with rhizosphere and rhizoplane of lentil and sesame at different growth stages

Density and diversity of Fusarium species and other fungi associated with rhizosphere and rhizoplane of lentil and sesame plants at three different growth stages were investigated. Sixteen species of Fusarium were isolated from rhizosphere (13 species) and rhizoplane (11) of both plants studied. In lentil, 11 species were recorded from its rhizosphere (9 species) and rhizoplane (8). Fusarium species associated with lentil rhizoplane gave highest number of propagules at the first stage of plant growth while the ones of Fusarium associated with the rhizosphere produced the highest number at the second stage of growth. F. solani was the most common in the three growth stages. In addition, of two growth stages, F. culmorum and F. tricinctum were isolated from the rhizosphere while F. nygamai and F. verticillioides from the rhizoplane. The other species were recorded from only one growth stage of lentil plant. In sesame plants, rhizosphere yielded nine Fusarium species while rhizoplane gave only six from the three stages investigated. Stage I of sesame rhizosphere possessed the highest colony forming units of Fusarium. As the case for lentil, F. solani was the most common species in sesame rhizospere and rhizoplane. F. verticillioides and F. nygamai (in three different growth stages) followed by F. oxysporum and F. tricinctum (in two growth stages) were recorded using the dilution-plate and/or soil-plate methods from sesame rhizosphere soils. Rhizoplane Fusarium species of sesame plants were isolated at the three different growth stages with almost equal number of colony forming units. F. poae came after F. solani in its frequency since it was recovered from two growth stages. Several of the isolated species are well-known as pathogens to many cultivated plants. To the best of our knowledge, three species are recorded here for the first time in Egypt from the rhizosphere (F. acutatum), rhizoplane of sesame plants (F. longipes) and from rhizosphere of both lentil and sesame and rhizoplane of lentil (F. nygamai).


INTRODUCTION
The terms rhizosphere and rhizoplane are now widely used by microbial ecologists and pathologists.Because of the widespread interest in the parasitic fungi attacking roots, numerous investigations have been made to characterize the fungus flora of root surface (Katznelson et al. 1948;Davey, Papavizas 1960;Srivastava, Mishra 1971;Abdel-Fattah et al. 1977;El-Hissy et al. 1980;Foster 1986;Campbell, Neher 1996;Mandeel 2002).Successful manipulation of rhizosphere and rhizoplane microorganisms to enhance biological disease control depends on knowledge of their ecological associations (Schroth, Hancock 1981;Mandeel, Baker 1991).
Lentil (protein-rich) and sesame (oilseed and food) are of the most important crops not only in Egypt but also in many other parts of the world (Bhatty 1988).However, In Egypt the areas under lentil and sesame cultivation tend to decrease due to the high infection levels by fungal diseases.Fungal diseases cause severe damage to roots, stems, leaves and pods of both lentil (Taylor et al. 2007) and sesame plants (Kolte 1985) of which root-rot and vascular wilt (caused by Fusarium oxysporum) are among the most important in Egypt in sesame (Hamdi et al. 1991) and lentil (Morsy 2005).
Since no reports are available on Fusarium species that might affect roots at different growth stages of lentil and sesame in Egypt, the present work aimed to survey Fusarium species and other fungal taxa from the rhizosphere and rhizoplane of lentil and sesame plants at three different growth stages.

MATERIALS AND METHODS
Rhizosphere sampling.Lentil (Lens culinaris Medik.) and sesame (Sesamum indicum L.) seeds were cultivated at the Botanical Garden of Botany Department, Faculty of Science, Assiut University.Roots of these plants were gently removed with superfluous soil layer at three different growth stages (stage I after 7 days, stage II after 45 days and stage III after 75 days from cultivation).The rhizosphere soils were placed in clean polyethylene plastic bags and transferred to laboratory for determining rhizosphere fungi.
Rhizoplane sampling.From the same plants and at the same stages, roots of lentil and sesame were collected as well as uprooted, dislodged from the adhering soil and directly transferred into clean polyethylene plastic bags and transferred to laboratory for determining rhizoplane fungi.
Isolation of rhizosphere Fusarium and other fungi of lentil and sesame plants.This group of fungi was isolated using the dilution-plate and soil-plate methods.
Dilution-plate method.The dilution-plate method was used to determine Fusarium and other fungi in soil as described by Johnson et al. (1959) and as employed in this laboratory by Moubasher and Abdel-Hafez (1978a, b) and could be summarized as follows: 1-Two g of roots with adhering soil particles were placed in weighed flask that contained 100 ml of sterile distilled water, thoroughly shaking for about 5 minutes.
2-One ml of the suitable rhizosphere soil suspension was transferred to each sterile Petri-dish and covered with melted but cooled dichloran chloramphenicol peptone agar (DCPA) (Andrews, Pitt 1986).For every rhizosphere sample, 5 agar plates were used.The plates were incubated at 25°C for 7-10 days and the developing fungi were counted, isolated and identified.The counts were calculated as colony forming units (CFU) per g of rhizosphere soil.The number of colonies for each fungus in each plate was multiplied by the dilution factor to obtain the number per g in the original soil sample, and then the average per 5 plates was calculated.
Soil-plate method.Soil-plate method adopted from Warcup (1950) was used to isolate rhizosphere fungi.Adhering or associated soil particles to the roots of tested plants were removed and 0.2 g of soil particles were transferred into the surface of each of DCPA agar plates.The plates were incubated at 25°C for 7-10 days.The developing fungi were counted, isolated, identified and thereafter calculated as CFU per g of dry soil.
Isolation of rhizoplane Fusaria and other fungi of lentil and sesame plants.The estimation of these fungi was carried out as practicised by Abdel-Hafez et al. (1990).The roots of plants were subjected to a series of washing with sterile distilled water.They were thoroughly dried between sterile filter papers, cut into segments (each about 1 cm) and 25 of these segments (5 per plate) were put on the surface of 5 DCPA agar plates.The plates were incubated at 25°C for 7 days and the developing colonies were counted, isolated and identified.The fungal counts were expressed as CFU per 25 segments of fresh roots.

Fusarium species and other fungi associated with rhizosphere soil of lentil plants.
Nine Fusarium species and ten genera of other fungi were identified from isolates of the three tested growth stages (stage I after 7 d, stage II after 45 d and stage III after 75 d from cultivation) using the dilution-plate and soil-plate methods (Tab.1).
The dilution-plate method revealed recovery of Fusarium CFU, that accounted at 33.33, 62.07 and 38.71% of total fungi taxa in the three stages, respectively, and the highest count was obtained in stage II.Seven species in addition to some unidentified species of Fusarium were recorded from the three tested growth stages (Tab.2).Fusarium solani was isolated from the every growth stage.F. culmorum and F. tricinctum were isolated from two growth stages while F. chlamydosporum, F. lateritium, F. nygamai and F. sporotrichioides were isolated from single growth stage.Other eight fungal genera were also recorded with Aspergillus P. Micheli ex Link as the only taxon isolated in all growth stages.The soil-plate method resulted in recovering Fusarium from the three tested growth stages, constituting 58, 60.82 and 23% of total fungi CFU, respectively and the highest count was obtained in stage II.Six species of Fusarium were recorded (Tab.2).Single species, F. solani, was isolated from two growth stages of lentil plants while the remaining five F. lateritium, F. nygamai, F. oxysporum, F. tricinctum and F. verticillioides were isolated from one growth stage of lentil plants (Tab.2).
Other nine fungal genera were also recorded with Aspergillus as the only taxon isolated in all growth stages.Species of Penicillium Link, Stachybotrys Corda, Stemphylium Wallroth and Trichurus Clem.were isolated from two growth stages of lentil plants (Tab.2).
Fusarium species and other fungi associated with rhizoplane of lentil plants.Fusarium fungi were isolated frequently from the plants in all three growth stages.They constituted 82.86, 58.97 and 72.09% of total fungi CFU, respectively.Eight species in addition to some unidentified species of Fusarium were recorded at the three growth stages (Tabs 1, 3).F. solani was isolated from the three growth stages of lentil plants.F. nygamai and F. verticillioides were recorded in two growth stages whereas F. culmorum, F. lateritium and F. oxysporum were recorded in only the first growth stage and F. subglutinans and F. udum in only the third stage (Tab.3).
Other four fungal genera were recorded at different growth stages of which Aspergillus fungi were isolated in all stages.
Fusarium species and other fungi associated with rhizosphere of sesame plants.Nine Fusarium species and eleven genera of other fungi were isolated from the three   growth stages investigated of sesame plants using the dilution-plate and soil-plate methods (Tabs 4, 5).
The dilution-plate method revealed the discovery of Fusarium from the three tested growth stages, constituting 52.17, 18.87 and 28.38% of total fungi in the three stages, respectively.The highest count was obtained in stage I (Tab.4).Seven species in addition to some unidentified species of Fusarium were recorded from rhizosphere soil at the three tested growth stages.F. solani was isolated from the three growth stages with its best count being found in stage I. F. oxysporum was isolated from two growth stages only.F. acutatum, F. poae, F. sambucinum, F, semitectum and F. tricinctum were isolated from one growth stage.
Other nine fungal genera were also recorded on this medium from the tested growth stages.Among them Aspergillus and Alternaria Nees were isolated from all growth stages.
The soil-plate method resulted in recovery of Fusarium from the three tested growth stages which constituted 82.81, 33.33 and 61.4% of total fungi CFU in the three stages, respectively with the highest count in stage I. Five species in addition to some unidentified species of Fusarium were recorded (Tab.4).F. nygamai, F. solani and F. verticillioides were isolated from the three growth stages of sesame plants.F. tricinctum was isolated from two growth stages while F. oxysporum was isolated only from one growth stage, comprising high percentages of total CFU.
Other six fungal genera were also recorded from the tested growth stages of which Aspergillus was isolated in all growth stages.Gliocladium Corda and Setosphaeria Leonard & Suggs were isolated from two growth stages.
Fusarium species and other fungi associated with rhizoplane of sesame plants.Fusarium was isolated from the rhizoplane of the three stages of sesame plant, comprising 76.67, 85.71 and 53.85% of total fungi, respectively.Its count varied between 21-24 CFU/25 sesame root segments and the highest count was obtained in stage II.
Six species in addition to some unidentified species of Fusarium were recorded (Tab.5).F. solani was recovered from the three stages with its highest CFU being found in stage II.F. poae was recorded in two growth stages, while F. chlamydosporum, F. longipes, F. oxysporum and F. verticillioides were recorded in one growth stage (Tab.5).
Other three fungal genera were also recorded at the three growth stages and Aspergillus species was the most common.
In total, 16 species of Fusarium were identified from rhizosphere (12 species using the dilution-plate and soil-plate methods), and rhizoplane (11 species using segment-plate method) of the two tested plants.F. solani was revealed in the three stages of both plants and its best count (CFU) was recorded during stage I or II.F. oxysporum was infrequently recovered from the three growth stages of the two investigated plants.In this respect, Li et al. (2012) isolated F. oxysporum, F. solani, F. proliferatum (Matsushima) Nirenberg and other 2 unidentified species from wilted sesame plants in China.In the studies of Fletcher et al. (1991) in New Zealand Fusarium avenaceum was reported as a major cause of collar rot and root rot of lentil while F. oxysporum, F. culmorum and F. acuminatum evoked disease symptoms but to a lesser extent than F. avenaceum.The last species influence was studied by Hwang et al. (2000) in Western Canada.
The remaining Fusarium species were encountered only from one or two stages.Some of these species were also recovered from the rhizophere and rhizoplane of some cultivated plants in Egypt such as cotton (Abdel-Hafez 1974), pea, tomato, maize and wheat seedlings (Moubasher et al. 1984), wheat (Abdel-Hafez et al. 1990, 2000) and sugarcane (Abdel-Hafez et al. 1995).Species of Fusarium and other fungal taxa reported earlier from Egypt as pathogens of lentil or sesame are shown in Table 6.
It is meaningful that some species were recorded only from the rhizosphere (F.acutatum, F. sambucinum, F. semitectum), rhizoplane of sesame plants (F.longipes) or both (F.poae), while others only on lentil plants from the rhizosphere (F.sporotrichioides), rhizoplane (F.subglutinans, F. udum) or both (F. culmorum and F. lateritium).It is also worthy to mention that F. acutatum is being recorded for the first time in Egypt from the rhizosphere and, F. longipes -from the rhizoplane of sesame plants, and F. nygamai -from the rhizosphere of lentil and sesame and rhizoplane of lentil plants.
Fungi other than Fusarium were also reported e.g., Aspergillus and Penicillium were commonly isolated from the rhizosphere and rhizoplane of both plants while others were reported only from one plant or from only rhizosphere or rhizoplane of one or both plants (Tab.1).To this group of fungi belong Alternaria, Cladosporium and Stemphylium are among referred to as the pathogens of lentil (Kaiser 1981).Stemphylium botryosum was reported to cause Stemphylium blight of lentil in many parts of the world including Egypt (Bayaa, Erskine 1998;Rahman et al. 2010).Those and other fungi taxa, namely member of Alternaria, Aspergillus (A. niger, A. flavus, A. terreus Thom), Botrytis Micheli, Chaetomium Kunze, Macrophomina phaseolina, Mucor Fresenius, Nigrospora Zimmermann, Penicillium, Phoma (Fries) Desmazieres, Rhizopus Ehrenb., Rhizoctonia solani, Stemphylium and Trichoderma Persoon were also isolated from lentil seeds in U.S. Pacific Northwest (Kaiser 1992) and Pakistan (Hussain et al. 2007;Rahim et al. 2010).The short literature survey of the pathogens recorded previously from lentil and sesame in Egypt is presented in Table 6.

CONCLUSIONS
Of the sixteen Fusarium species isolated during the current investigation, only three, namely F. oxysporum, F. solani and F. verticillioides were recorded, from the rhizosphere and rhizoplane of both plants.However, only F. solani was recorded at the three stages of both plants.Of the species recorded, some are known as pathogenic to both plant species, and others are recorded for the first time from lentil or sesame in Egypt.Of fungi other than Fusarium, only Aspergillus and Penicillium species were recorded from both rhizosphere and rhizoplane of both plants.
Abbreviations: CFU: Colony forming units per gram of soil (calculated out of 5 agar plates in each case); CFU%: calculated per total colony forming units of all fungi;Stage I: after 7 days, Stage II: 45 d, Stage III: 75 d from a cultivation.
Abbreviations.CFU: Colony forming units per gram soil (calculated out of 5 agar plates in each case); CFU%: calculated per total colony forming units of all fungi;Stage I: after 7 days, Stage II: 45 d, Stage III: 75 d from a cultivation.

Table 1
Summarized data showing the source of isolation of the pathogens +Table2Colony forming units (CFU) of Fusarium species and other fungi associated with rhizosphere soil cultivated with lentil at different growth stages on DCPA at 25°C using dilution-plate and soil-plate methods

Table 3
Colony forming units of Fusarium species and other fungi taxa associated with rhizoplane of lentil plants at three growth stages Abbreviations: CFU: Colony forming units (out of 25 lentil root segments); CFU%: calculated per total colony forming units of all fungi; Stage I: after 7 days, Stage II: 45 d, Stage III: 75 d from a cultivation.

Table 4
Colony forming units of Fusarium species and other fungi taxa associated with rhizosphere soil cultivated with sesame at different growth stages on DCPA at 25 °C using dilution-plate and soil-plate methods

Table 5
Colony forming units of Fusarium species and other fungi associated with rhizoplane of sesame plant at three different growth stages Abbreviations.CFU: Colony forming units (out of 25 sesame root segments); CFU%: calculated per total colony forming units of all fungi.Stage I: after 7 days, Stage II: 45 d, Stage III: 75 d from a cultivation.

Table 6
Fungal species reported from Egypt as lentil and sesame pathogens by other authors