Abstract: Powdered shoot extract of Launaea procumbens, a tropical ruderal and agrestal weed, inhibited egg hatch and caused mortality of Meloidogyne javanica juveniles in vitro. However, ethanol extract of L. procumbens did not inhibit radial growth of root-infecting fungi including Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani in vitro. Soil amendment with powdered shoot of L. procumbens markedly reduced root-knot infection caused by M. javanica in mungbean. Population densities of M. javanica were significantly lower in soil amended with 5.0% L. procumbens while a 2.5% amendment did not produce significant reduction in the nematode populations in soil. Whereas low dosage (2.5%) of L. procumbens significantly enhanced plant growth, high dosage (5%) reduced fresh shoot and root weights of mungbean indicating allelopathic effect. Soil amendment with L. procumbens resulted in marked changes in fungal community structure and composition. Fungi like Fusarium semitectum and a sterile fungus (red pigmented) were exclusively isolated from L. procumbens amended soils. On the other hand, all the fungal species isolated from L. procumbens amended soils were also present in unamended soils. Soil amendments with L. procumbens also altered fungal community structure in the root tissues of mungbean. Both general diversity and equitability of fungal community at 2.5% L. procumbens increased appreciably over the controls but at 5% dosage substantially decreased compared to controls, substantially though species richness declined at both the dosages. Dominance concentration followed an opposite trend to that of general diversity.
Introduction
The soil-borne root-knot nematode and root-infecting fungi play a major role in the development of root-rot disease complexes on many important field and horticultural crops that often results in reduced growth and even death of plants. The problem of such pathogens is more serious in Pakistan than in most developing countries. Firstly, because of tropical climatic conditions, fungi and nematode proliferate rapidly throughout the year. Secondly, sandy soils particularly those which are irrigated have conditions conducive to the growth and survival of the pathogens. There exists unquestionable potential for managing plant diseases incited by soil-borne phytopathogens and increasing crop productivity with the application of botanical toxicants to the soil. The interest in the use of organic amendment for the management of plant-parasitic nematodes has recently been intensified because of the phasing out of the chemical pesticides from the market of the developed countries in the year 2005. It is likely that the farmers in the developing countries will consume large quantities of such chemical pesticides. Therefore, effective alternative methods to combat soil-borne root-infecting fungi and root-knot nematode are urgently needed.
Allelopathy is a plant-plant or plant-microorganism biochemical interaction (Rice, 1981). Many weeds interfere with the crop plants through production of chemical substances (allelochemicals) that inhibit their growth and development. The allelochemicals produced by plants are varied including phenolic acids, terpenes, terpenoids, glycosides, alkaloids and flavonoids (Whittaker and Feeny, 1971; Mandava, 1985; Blum, 1996). Besides, a number of secondary metabolites of plants are toxic to nematodes and fungi. Tegetes spp., produce α-terthienyl whereas Crotalaria spp. produce mono-crotaline, both of which have nematicidal qualities (Fassuliotis and Skucas, 1969; Gommers and Bakker, 1988). Similarly, some toxic compounds synthesized by Lantana camara cause substantial mortality of Meloidogyne javanica juveniles in vitro (Ali et al., 2001). In another study, Shaukat and Siddiqui (2001) showed that L. camara also possesses antifungal agents, which inhibit radial growth of root-infecting fungi including Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani in vitro. It was demonstrated that when powdered shoot material was added to the soil, ability to cause root-infection by such fungi was greatly abated. Shaukat et al. (2001) demonstrated in vitro nematicidal activity of the powdered shoot extract of Argemone mexicana that is known to produce a number of phenolic acids including salicylic acid.
Launaea procumbens, a tropical ruderal and agrestal weed, grows abundantly in waste grounds, vacant lots, lawns and abandoned and cultivated fields in Southern Sind. Shaukat et al. (2003) showed that shoot extract of L. procumbens caused seed germination and early seedling growth of four test species including mustard, bulrush millet, corn and spinach. In the same study, these authors showed that L. procumbens produces compounds like salicylic acid, vanillic acid, syringic acid, 2-methyl-resorcinol and gallic acid (Shaukat et al., 2003) which is likely to be responsible for the inhibition of plant growth. Whereas allelopathic potential of L. procumbens is known, nematicidal and antifungal activities of this weed have remained largely unexplored.
The aim of the present investigation was to examine I) the nematicidal, allelopathic and antifungal activity, if any, of L. procumbens in vitro, ii) to test the nematicidal and allelopathic potential of the weed in pot cultures under glasshouse conditions and iii) to investigate the fungal community structure of the rhizosphere, in particular, community diversity as influenced by the weed residues.
Materials and Methods
Plant material and preparation of shoot extract
L. procumbens was collected from a waste ground in Malir, Karachi
and its shoot material was air dried and powdered in an electric grinder. The
powdered shoot material (50 g) was soaked in 500 ml sterile distilled water
and left for 72 h at room temperature. The extract was filtered through two
layers of Whatman No.1 filter paper and kept at 6°C prior to use. This was
called stock solution and from it 50% stock solution was obtained using sterile
distilled water.
Nematode and fungal inoculum
Meloidogyne javanica (Treub) Chitwood was obtained from pure cultures
maintained on roots of eggplants (Solanum melongena L.). The entire root
system was dipped in water and soil was removed gently without detaching egg
sacs. Eggs were extracted by vigorous shaking of infested roots in a 1% sodium
hypochlorite solution for 3 min. The resulting suspension was then passed through
a range of different mesh sieves. The eggs collected on a fine sieve (38 μm)
were washed in tap water to remove all traces of sodium hypochlorite before
use. Hatched juveniles of M. javanica were obtained by placing the eggs
in sterile distilled water for 3-5 days at 28°C. The inoculum was used for
laboratory and glasshouse tests.
Three root-infecting fungi were tested. Macrophomina phaseolina was isolated from infected bean (Phaseolus vulgaris L.) roots of plants growing at Ghulamullah, 62 km east of Karachi while Rhizoctonia solani and Fusarium solani respectively, were isolated from infected tomato and brinjal roots obtained from Mirpur Sakro, 69 km east of Karachi. The fungi were routinely cultivated on potato dextrose agar (PDA) plates supplemented with appropriate quantities of penicillin and streptomycin sulphate.
Effect of shoot extract on egg hatch of M. javanica
To study the effects of the weed extract on egg hatching of M. javanica,
two medium sized egg masses with 2 ml of the aqueous shoot extract of the weed
(L. procumbens) were transferred into a 1 cm diameter cavity glass slide.
The egg masses placed in sterile distilled water served as controls. Each treatment
was replicated three times and the cavity glass slides were randomized on laboratory
bench. The numbers of hatched juveniles were counted after 48 h. The egg masses
were then transferred into cavity glass slides containing 2 ml sterile distilled
water to ascertain whether the egg masses kept in the culture filtrate had been
temporarily or permanently inactivated. The juveniles were counted again after
a further 48 h period. The experiment was performed three times.
Effect of shoot extract on mortality of M. javanica juveniles
To study the effects of aqueous extract of the weed on mortality of M.
javanica, two ml of each filtrate were poured in a glass cavity slide and
about 30-40 second stage juveniles of M. javanica placed in each glass
slide. Juveniles kept in freshly prepared liquid medium served as controls.
Treatments were replicated three times and dead nematodes in each cavity slide
were counted after 24 and 48 h. The nematodes were considered to be dead when
they did not move on probing with a fine needle. The experiment was conducted
twice.
Preparation of ethanolic extract and its antifungal activity
Fresh shoots (50 g) of L. procumberns were soaked in ethanol (100
ml) and disintegrated in a homogenizer. After storing for 2 weeks, ethanolic
extract was filtered through 2 layers of Whatman No.1 filter paper. The ethanolic
extract was dried in a rotary vacuum evaporator under reduced pressure at room
temperature (30°C). The gummy substance so obtained was weighed and dissolved
in ethanol. To determine the antifungal activity, the ethanolic extract (10
mg ml-1) of L. procumbens was impregnated on a 5 mm diam disc
of Whatman No. 1 filter paper at 10 μl disc-1 and placed 5 mm
inside of the 9-cm-diam. Petri plates containing Czapek’s Dox Agar (CDA)
medium, pH 7.2. Disc dipped in ethanol served as control and was placed apart
from the disc containing ethanolic extract of L. procumbens. A 5-mm-diam.
disc of the test fungus was placed at the center of the Petri plate. There were
four replicates for each test fungus and plates were incubated at room temperature
(30°C). Distance covered by the fungus and zone of inhibition (if any) was
measured after 7 days for M. phaseolina and R. solani and 10 days
for Fusarium solani as this fungus is slow growing.
Nematicidal, antifungal and allelopathic responses of Launea procumbens
in mungbean
The soil (sandy loam; pH 7.8; moisture holding capacity 38%) was obtained
from Crop Disease Research Institute, Karachi University campus. The soil was
naturally infested with 6 species of plant-parasitic nematodes and some free-living
nematodes. The air-dried soil was passed through a 2 mm mesh sieve to discard
non-soil particles. The experiment consisted of 2 treatments: non-amended soil
planted with mungbean and L. procumbens-amended soil planted with mungbean
The experiment was set up as a randomized complete block design with 4 replications.
The soil was thoroughly mixed with L. procumbens at 2.5 or 5.0% w/w and
filled in 8-cm-diam. plastic pots at 400 g pot-1. The pots with soil
were placed in a glasshouse (18-26°C) and kept moist for 2 weeks. Subsequently,
eight surface sterilized mungbean seeds were sown in each pot and following
germination only four seedlings were retained pot-1. One week after
emergence, 2000 freshly hatched juveniles of Meloidogyne javanica were
added by making 4 holes in the soil around the seedlings in the pots with mungbean.
The soil in each pot was sprinkled with 200 ml of sterile distilled water each
day. The experiment was terminated 45 days after nematode inoculation and plant
height, fresh weight of shoot and root, number of galls induced by M. javanica
and nematode populations in the soil were estimated (see below).
Isolation and identification of fungi from rhizosphere
At harvest, one mungbean plant was randomly chosen from each replicate pot
to study the rhizosphere microfungi. The roots were excised and weighed after
the excess soil had been shaken-off. The roots were then shaken vigorously in
a test tube containing sterile distilled water, blotted dry and reweighed. A
serial dilution of the soil suspensions was prepared and was tested for the
enumeration of fungi. A 0.5 ml aliquot from 102 and 103
dilutions was plated onto CDA medium, supplemented with penicillin (100,000
units l-1) and streptomycin sulphate (0.2 g l-1) to avoid
bacterial contamination. After incubation at 28°C the plates were examined
for total fungal counts. Most isolates were obtained after a few days of incubation,
but plates were checked over several weeks to allow isolation of slow-growing
fungi. Developing fungal colonies were sub-cultured into pure isolates and identified
by their microscopic morphology using standard mycological literature (Booth
1971; Domsch et al., 1980; Thom and Rapper, 1945).
Isolation of fungi from roots
The roots of all plants (including the one which was tested for the isolation
of rhizosphere fungi) were cut into small segments (5 mm) and after surface
sterilization in 1% Ca(OCl)2 for 3 min, 5 such segments were plated
onto potato dextrose agar (PDA) plates supplemented with penicillin (100,000
units l-1) and streptomycin sulphate (0.2 g l-1). The
plates were incubated at 28°C for one week and emerging fungi from each
root segment were identified. Colonization percentage was determined by using
the following formula:
Root-knot development and nematode soil populations
The numbers of galls induced by M. javanica on the entire root system
were counted with the aid of a hand lens. For nematode counts, 100 cm3
soil aliquots were incubated for 72 h using a modified Baerman funnel technique
(Rodríguez-Kábana and Pope, 1981).
Statistical analysis
Data were subjected to one-way analysis of variance (ANOVA) followed by
the least significant differences test (LSD) or Duncan’s multiple range
test using STATISTICA software (1995, ver. 5.0; StatSoft Inc., Tulsa, Oklahoma,
U.S.A.). Fungal rhizosphere populations were transformed to log10
(x+1) before the analysis.
Diversity measurement
Species diversity is an important parameter of natural or organized community
and several diversity indices have been proposed (Magurran, 1988). Diversity
indices represent a useful means to quantify community diversity and have been
instrumental in revealing the impact of biocontrol inoculants on resident population
assemblages (Natsch et al., 1997). Several diversity indices were employed
to compare treatment effects. Various diversity measures estimate different
aspect of community structure. The general species diversity of the fungal communities
was measured by the generally accepted Shannon-Wiener information theory function:
where H’ is the general species diversity and pi the proportion of total number of cfu for fungi or counts for nematodes, N belonging to the its species (Shannon and Weaver, 1963). The variance of general diversity var (H’) was calculated in accordance with Magurran (1988), as follows: Var (H’) = ∑pi (log pi)2 – (∑ pi log pi)2/N + (S-1)/2N2.
Dominance concentration (complement of diversity) was measured by using Simpson’s index (Southwood and Henderson, 2000) as: D = ∑ {[ni(ni-1)]/[N(N-1)]} in which ni = number of cfu for fungi or counts for nematodes. The general diversity incorporates two components of diversity: species richness, which expresses the number of species (S) as a function (ratio) of the total number of individuals (N) and equitability that measures the evenness of allotment of individuals among the species (Magurran, 1988). The equitability component of diversity and its variance were measured in accordance with Pielou (1975): J’ = H’/H’max. The equitability index J’ is the ratio between observed diversity (H’) and maximal diversity (H’max). Variance of equitability was estimated as: Var (J’) = Var (H’)/(log S)2.Species richness was calculated in accordance with Menhinick (1964) as d = S/√ N, where S equals the number of species and N the total number of individuals (colony counts).
Results
Effect of shoot extract on egg hatch of M. javanica
Shoot extract of Launaea procumbens significantly inhibited in
vitro egg hatching of Meloidogyne javanica (P<0.01) compared to
the controls (Table 1). The inhibitory effect was not circumvented
even after transfer of eggs from shoot extract to distilled water.
Effect of shoot extract on mortality of M. javanica juveniles
At both the time periods (24 and 48 h) shoot extract of L. procumbens
caused significantly (P<0.001) greater mortality of Meloidogyne javanica
compared to water controls (Table 2). The nematicidal effect
was substantially more pronounced at 48 h compared to that at 24 h.
Effect of L. procumbens extract against root-infecting fungi
When the effect of shoot extract of L. procumbens was tested against
three different root-infecting fungi Rhizoctonia solani, Fusarium
solani and Macrophomina phaseolina it was observed that the fungal
growth in Petri plates extended and even overgrew the disc impregnated with
L. procumbens extract (data not presented).
Effect of L. procumbens on the development of root-knot infection
and the growth of mungbean
Soil amendment with L. procumbens at both the dosages markedly (P<0.05)
reduced galling intensity due to M. javanica compared to the controls
(Table 3). No significant difference between two dosages of
L. procumbens was observed with respect to galling. Population densities
of M. javanica were significantly (P<0.05) lower in soil amended with
5.0% L. procumbens while a 2.5% amendment failed to reduce nematode populations
in soil. When compared to the controls, soil application of L. procumbens
resulted in a significant (P<0.05) increase in plant height at 2.5% but
not at 5.0% L. procumbens. Shoot weight was significantly (P<0.05)
enhanced at 2.5% while a significant (P<0.05) reduction occurred in shoot
and root weights at 5% L. procumbens.
Effects of L. procumbens on fungi in the rhizosphere
A total of 16 microfungal species comprising of 10 genera were isolated
from the rhizosphere of mungbean amended with or without L. procumbens
(Table 4). The numbers of fungal species were significantly
higher in soil amended with L. procumbens compared with unamended soil.
Similarly, colony counts of the fungi were markedly higher in L. procumbens
amended soils. When two dosages of L. procumbens were compared, cfu
of the fungi including
| Table 1: | Effects of Launea procumbens on egg hatching of Meloidogyne javanica |
| aAfter a 48 h hatching period in culture filtrate, the egg masses were transferred to sterile distilled water | |
| Table 2: | Effects of aqueous extract of Launaea procumbens shoot extract on mortality of Meloidogyne javanica |
| Table 3: | Effects of Launaea procumbens on root-knot development due to Meloidogyne jacanica and growth of mungbean plants |
| Means with the same letter are not significantly different at P<0.05 | |
| Table 4: | Effect of soil amendment with or without shoot powder of Launaea procumbens on soil fungal community structure expressed as log10 (x+1) in mungbean |
| Table 5: | General diversity H’, equitability (J’), species richness (d) and dominance (D) of the fungal communities affected by soil amendment with or without Launaea procumbens in mungbean rhizosphere. Var (H’)= ariance of H’; Var (J’) = variance of J’ |
| Table 6: | Percent colonization of the fungi isolated from the mungbean roots growing in soils amended with L. procumbens |
Acremonium butyric, Aspergillus flavus, A. niger A. quadrilinatus, Macrophomina phaseolina and Penicillium brefeldianum were markedly higher in soil amended with 2.5% L. procumbens. Colony numbers for other fungi were higher in soil amended with 5.0% L. procumbens. Fusarium semitectum and a sterile fungus (red pigmented) were exclusively isolated from L. procumbens amended soils. None of the fungi were specifically isolated from unamended soils and all fungi isolated from L. prcumbens amended soils were also recorded from unamended soils
Effects of L. procumbens on the diversity of rhizosphere fungi
Both general diversity and equitability of fungal community at 2.5% L.
procumbens increased appreciably over the controls but at 5% dosage decreased
substantially compared to controls (Table 5). On the other
hand, species richness declined at both the dosages. Dominance concentration
as measured by Simpson’s index followed an opposite trend to that of general
diversity.
Effects of L. procumbens on root colonization by fungi
The fungi isolated from the roots of mungbean included Aspergillus
sp., Fusarium solani, Mycelia sterilia, Macrophomina phaseolina
and Rhizoctonia solani (Table 6). Aspergillus
sp. and Fusarium solani exhibited greater colonization percentage of
roots in amended soils (2.5 and 5% L. procumbens) compared to the controls,
whilst Mycelia sterilia occurred only in the amended soils. On the other hand,
Macrophomina phaseolina and Rhizoctonia solani colonized mungbean
roots to a lesser extent in the amended soils compared to non-amended controls.
Discussion
Soil amendment with Launaea procumbens caused significant reduction of M. javanica population densities in soil, nematode penetration and subsequent root-knot infection in mungbean. Soil amendments with L. procumbens also resulted in marked changes in fungal communities both in the rhizosphere and inner root tissues. Understanding how L. procumbens amendment controls root-knot infection and what soil factors regulate activity is critical for reducing the application rates needed and improving the efficacy of the organic amendment. Several mechanisms of disease suppression can be involved including compounds toxic to nematodes (Ali et al., 2001; Shaukat and Siddiqui, 2001a) changes in microbial communities suppressive to nematode (Hallmann et al., 1999; Shaukat and Siddiqui, 2001b) and stimulation of the activity of biological control organisms (Siddiqui et al., 1999). Observed reduction in nematode population densities in the soil and root suggests that toxic compound in the organic amendment generated following incorporation, could be involved. Disease reduction due to toxic compounds is the easiest mechanism to assess, providing one can determine the viability of a pathogen in the soil.
Since most potential fungicides are highly toxic to human beings and produce environmental hazards, application of botanical toxicants in the soil provides an effective alternative means to control fungal pathogens. However, the majority of soil fungi are nonpathogenic and a large number of these may even be beneficial to plants and/or contribute positively to ecosystem functioning. Indeed, nonpathogenic saprotrophic microfungi perform key ecological role in the soil ecosystem through decomposition of organic matter, nutrient cycling, natural control of plant pathogens and a myriad of other functions (Cooke and Rayner, 1984; Curl and Truelove, 1986; Dix and Webster, 1995). In this context, it is surprising that saprotrophic rhizosphere fungi have been largely neglected asnon-target, beneficial resident microorganisms potentially affected by specific organic amendment, especially when the latter produce antifungal and/or nematicidal metabolites with a relatively broad range of action.
It is interesting to note that species of Aspergillus and Rhizopus stolonifer were isolated relatively in large numbers from the amended soils. Enhanced populations of these fungi in the rhizosphere following soil amendment with L. procumbens could be of significant advantage. Penicillium and Fusarium are well documented as decomposers of celluloses and hemicelluloses(Domsch et al., 1980). The ability of certain strains of saprotrophic Fusarium solani strains to protect plants against pathogenic fungi through competition, parasitism, antagonism and/or induced resistance is well known (Alabouvette and Steinberg , 1995; Chet et al., 1997; Fuchs et al., 1997; Amer-Zareen et al., 2001). Species of Aspergillus (Siddiqui et al., 2001) and non-pathogenic Fusarium (Amer-Zareen et al., 2001) are also known to suppress root-knot nematode populations and their infectivity.
Species of Fusarium and Rhizoctonia are common inhabitants of most agricultural fields in Pakistan and are considered as the most devastating pathogens causing severe losses in economically important crops including mungbean. In the present study, populations of F. oxysporum and F. solani in the rhizosphere markedly increased with time and that amended soils supported larger populations of both the fungi. In contrast, populations of R. solani were relatively higher in non-amended soils. Interestingly, inner root colonization by F. solani and R. solani reduced in amended soils compared with those of non-amended soils while F. oxysporum did not colonize mungbean roots in any of the soils. It is possible that the release of phytoalexins in response to colonization by Fusarium spp. and Rhizoctonia solani could have been a contributing mechanism in reducing fungal penetration and colonization. Furthermore, soil harbors a variety of microorganisms including saprophytic bacteria that are known to induce systemic resistance in plants against pathogenic fungi and nematodes.
Isolation of fungi from the rhizosphere of mungbean yielded a broad fungal spectrum dominated by genera and species rather widespread and frequently found in agricultural soils, rhizospheres and roots of crop plants. This fungal spectrum overlaps the one obtained by Hong (1969) and Girlanda et al. (2001) who found that rhizosphere fungi protect cucumber seedlings against damping-off caused by Fusarium oxysporumf. sp. cucumerinum. No oomycetes (Pythium and Phytophthora) were isolated in this study, despite the fact that they can grow on the laboratory medium used. This is in accordance with the fact that disease pressure is usually low in the experimental field of the Department of Botany, University of Karachi. Furthermore, cool climate favours the growth and survival of these fungi in the soil, which during mungbean cultivation season was not available. It is also possible that oomycetes were present at population levels too low to be detected, or perhaps they were not competitive enough on the culture plates.
While changes in the soil fungal community following L. procumbens amendment were anticipated, interestingly, the endophytic fungal community of mungbean was also considerably influenced quantitatively. Five fungal species were recorded as endophytes and only one was specifically present in the amendments. However, quantitative differences in controls and amendments were amply prominent. These results suggest that endophytic fungi are predominantly recruited from the rhizosphere where they presumably use wounds and natural opening to enter the roots. Lytic enzymes produced by these fungi might also contribute to more efficient penetration and colonization. Endophytes colonize the same root tissues as sedentary plant-parasitic nematodes therefore, this association of endophytic fungi with nematodes throughout the nematode life cycles makes these fungi excellent candidates for biocontrol strategies. Nevertheless some of these fungi may cause hypersensitive reactions in plants.
In the present study, whereas soil amendment with powdered shoot of L. procumbens at low dosages (2.5% w/w) enhanced shoot growth of mungbean, a high concentration (5% w/w) invariably reduced shoot and root growth of mungbean plants. The inhibitory effect of L. procumbens is presumably the result of the presence of phenolic compounds including salicylic acid, vanillic acid, syringic acid, 2-methyl resorcinol, gallic acid and two unknowns in the shoot of L. procumbens (Shaukat et al., 2003) due to which it expresses its allelopathic activity. The toxic effects of the phenolic compounds on seed germination and plant growth have been previously reported (Blum, 1996; Inderjit, 1999; Burhan and Shaukat, 2000). However, the presence of allelopathic secondary metabolites other than phenolic compounds in L. procumbens cannot be ruled out. The development of microbial populations in response to high dosages of L. procumbens powdered shoot could constitute another factor that impaired plant growth.
Organic by-products are a source of energy and nutrients and when applied to soil change its biological, physical and chemical properties. Potential benefits from use of these soil amendments include (1) suppression of disease (2) improved soil physical characteristics (3) increased soil microbial diversity/population size (4) delivery of biological control agents or plant growth-promoting rhizobacteria (5) use for so called waste by-products. Potential risks include contamination of the environment and detrimental effects on plant growth and the growth of beneficial microorganisms such as plant growth-promoting rhizobacteria and mycorrhizal fungi. We must ensure that agricultural soils do not become a refuse centre for organic trash. If there is to be a role for amendments as a viable disease control strategy, we need to determine where and how amendments affect diseases, manipulate amendments and their dosages in the soil for maximum efficacy and provide farmers access to skilled technical assistance. Presently in the developing countries like Pakistan, organic amendments are not widely used to control plant diseases because of the lack of available information of the impact of such toxicants on pathogens. We believe that use of organic amendments will become a viable disease control strategy in Pakistan for mungbean and other high value crops. In other areas, the potential for use of amendments is even greater where the cultivation of higher value crops, options for double and triple cropping and the occurrence of soils of extreme alkalinity or acidity exist.
Acknowledgments
Technical assistance provided by Syed Azhar Ali and his staff at Haider Ali Farm, Gharo and Maria Hamid, University of Karachi, is acknowledged with gratitude.