Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Additive inheritance of resistance to pod rot caused by Phytophthora palmivora in cocoa

Summary Quantitative inheritance of resistance to Phytophthora pod rot (Ppr) was studied in cocoa hybrid progeny from 12 Trinitario x Amazonian crosses and their reciprocal crosses. The crossing scheme was similar to a factorial design. Disease was assessed by the number and percentage of infected pods on each tree. Highly significant differences due to general combining abilities (GCA) were obtained for all characters, except for the GCA of Trinitario on total pod production. Differences for specific combining ability (SCA) were not significant for all characters. There were no significant differences between reciprocal crosses. The Trinitario clone K82 provided the only source for the hybrid progenies of strong Ppr resistance to the hybrid progenies, while K20 provided moderate resistance. Other parental clones — KA2-101, KA5-201, KEE 2, KEE 5, and KEE 52 — produced progenies which were susceptible to Ppr. It is evident that resistance to Ppr in cocoa is inherited additively. Maternal and cytoplasmic effects were assumed to have no influence on inheritance of resistance. It is also concluded that resistance to Ppr of the kind shown by K82 is likely to be horizontal resistance. Breeding for high-yielding cultivars combined with Ppr resistance is the most effective way of controlling Ppr of cocoa on the crops of growers with small holdings in Papua New Guinea.     

Defective zoospore encystment and suppressed cyst germination of Phytophthora palmivora caused by transient leaching treatments

The behaviour of encysting zoospores of Phytophthora palmivora during leaching conditions was studied. Zoospores encysted and germinated successfully on polycarbonate membranes after mechanical agitation. Transient (10 min) leaching treatments with nutrient-free buffer underneath the membranes resulted in abnormal encystment and poor germination. The disruption was greatest when leaching was applied during the first minutes after start of encystment and not observed after 20 min. The early sensitivity of cells to leaching coincided with the period when alkali-resistant cell walls were formed (2 – 6 min after mechanical agitation). Effects of calcium and organic nutrients on encystment during leaching and germination after these treatments were studied. The disruption of encystment by early leaching treatments, but not the suppression of cyst germination, was overcome by adding calcium chloride during mechanical agitation of zoospores. Leaching with calcium containing buffer resulted in suppressed cyst germination as was the case with buffer alone. Leaching with 0.1 % peptone containing buffer promoted consistently high encystment and germination. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro expression of partial resistance toPhytophthora palmivora by shoot cultures of papaya

Shoot apices ofCarica papaya were multiplied in vitro on solidified nutrient media supplemented with -naphthyl-acetic acid and 6-benzylaminopurine. The micropropagated shoots were inoculated in vitro, through a stem wound, with a sporangial suspension (1.2×10⁴ sporangia ml^-1) ofPhytophthora palmivora. The symptoms exhibited by the shoots in vitro were similar to those described previously for infection of the whole plant in the field. The time taken for the host tissue to become brown and to wilt and the time to sporulation of the pathogen were all recorded for each shoot of four varieties of papaya challenged with each of ten isolates ofP. palmivora. Significant differences were observed between host-pathogen combinations for these variables and host-specificity was detected amongst the isolates ofP. palmivora. The time taken for the shoot to wilt was positively correlated with the time to sporulation of the isolated but both these variables were negatively correlated with the time to browning of the shoot. In vitro selection for disease resistance will be useful during breeding programmes involving papaya genotypes which are maintained through clonal propagation.     

A new oil-based formulation of Trichoderma asperellum for the biological control of cacao black pod disease caused by Phytophthora megakarya

In African cacao-producing countries, control of cacao black pod disease caused by Phytophthora megakarya is a priority. Introducing biological control agents as part of a P. megakarya control strategy is highly desirable, especially in a perspective of pesticide reduction. Trichoderma species are among the most used biological control agents. In Cameroon, Trichoderma asperellum formulated in wettable powder has produced positive effects against this disease. However, with this type of formulation, shelf-life and persistence of conidia on pods are limited. Our study therefore sought to develop a new T. asperellum formulation that would be more effective and better suited to the conditions of field application by small-scale producers in Cameroon. We selected a soybean oil-based oil dispersion, in which the half-life of the conidia reached 22.5 weeks, versus 5 weeks in aqueous suspension. Tested on detached pods, the formulation completely inhibited the development of the disease. When sprayed in the field on cacao clones highly sensitive to P. megakarya, the formulation resulted in 90% protection of treated pods after 1 week, and 50% after 3.2 weeks. The formulations exercised a measurable effect for up to 7 weeks, versus 2 weeks in the case of an aqueous conidial suspension and 5 weeks for that of a conventional fungicide (Kocide). Trichoderma asperellum formulated in oil dispersion has therefore great potential for the control of cacao black pod disease with less recourse to synthetic fungicides. Moreover, this formulation is well adapted to the types of sprayers used by small-scale cacao producers in Cameroon.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

The effect of biofungicide Mycostop on Ceratocystis radicicola, the causal agent of black scorch on date palm

In vitro studies were conducted to assess the efficacy of the biofungicide Mycostop on the fungus Ceratocystis radicicola (= Ceratostomella radicicola), which causes black scorch on date palm (Phoenix dactylifera) and the effect salinity on the growth of Streptomyces griseoviridis, the activecomponent of Mycostop. C. radicicola wasisolated from roots of diseased date palm inlandscape settings, nurseries, andplantations. In vitro, the effectiveconcentration (EC₅₀) of Mycostop thatreduced the mycelial growth by 50% was 0.35 g/l of PDA. Mycostop at the rate of 0.35 g/lor greater reduced spore germination,plasmolyzed germlings and reduced sporulationof C. radicicola. In essence it reducedthe inoculum potential of C. radicicola. In sterile soil treated with Mycostop, rootpieces of date palm inoculated with C.radicicola were less necrotic than those inuntreated soil. Streptomycesgriseoviridis in Mycostop did not grow on PDAamended with 5.0% NaCl (–4.28 MPa), but thenumber of colony formed units on PDA amendedwith 1.0 to 4.0% NaCl was not significantlydifferent from a factor that was considered ifMycostop is to be used to manage black scorchin Kuwait. The observations showed the effectsof Mycostop on C. radicicola inlaboratory studies but its efficacy on blackscorch disease of date palms in nurseries andplantations is yet to be determined.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

Biological control of Phytophthora blight in red pepper (Capsicum annuum L.) using Bacillus subtilis

Phytophthora blight is one of the most important devastating diseases of red pepper plants. Forty-one bacterial isolates were obtained from rhizosphere soil and subsequently tested for antagonistic activity under in vitro and in vivo conditions. Among the 41 isolates tested, 12 exhibited a maximum antagonistic activity in dual culture assay. These 12 isolates were further screened for disease suppression on red pepper plants in both natural and greenhouse conditions. All the antagonists showed varying levels of antagonism, whereas the isolates R33 and R13 exhibited the maximum (86.8 and 71%) ability to reduce the disease severity in in vivo conditions. Based on the 16S rDNA sequencing, the most effective isolates were identified as Bacillus subtilis. In addition, the isolates were also screened for siderophores, hydrogen cyanide and hydrolytic enzymes. Further, the isolates increased the root and shoot length of the red pepper, which is an added advantage of the isolates while performing the desired function.     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

RFLP mapping of resistance to the blackleg disease [causal agent,Leptosphaeria maculans (Desm.) Ces. et de Not.] in canola (Brassica napus L.)

We report the tagging of genes involved in blackleg resistance, present in the French cultivar Crésor of B. napus, with RFLP markers. A total of 218 cDNA probes were tested on the parental cultivars Crésor (resistant) and Westar (susceptible), and 141 polymorphic markers were used in a segregating population composed of 98 doubled-haploid lines (DH). A genetic map from this cross was constructed with 175 RFLP markers and allowed us to scan for specific chromosomal associations between response to blackleg infection and RFLP markers. Canola residues infested with virulent strains of Leptosphaeria maculans were used as inoculum and a suspension of pycnidiospores from cultures of L. maculans, including the highly virulent isolate Leroy, was sprayed to increase disease pressure. QTL mapping suggested that a single chromosomal region was responsible for resistance in each of the four environments tested. This QTL accounted for a high proportion of the variation of blackleg reaction in each of the assays. A second QTL, responsible for a small proportion of the variation of blackleg reaction, was present in one of four year-site assays. A Mendelian approach, using blackleg disease ratings for classifying DH lines as resistant or susceptible, also allowed us to map resistance in the region of the highly significant LOD scores observed in each environment by interval mapping. Results strongly support the presence of a single major gene, named LmFr ₁ controlling adult plant resistance to blackleg in spring oil-seed rape cultivar Crésor. Several RFLP markers were found associated with LmFr ₁.     

Characterization of the metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas (Pisum sativum L.)

The metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas, was detected by thin layer chromatography (TLC) and was analyzed for its chemical and pathogenic characteristics. One blue dot was detected using 254nm UV light on TLC plate, and a spray of rho-anisaldehyde (110 degrees C, 30 min) also produced a blue dot. The solvent systems used for TLC analysis were ethyl acetate/water/acetone (5/2/5), chloroform/methanol/glacial acetic acid (19/10/2), toluene/ethyl acetate/90% formic acid (6/3/1), diethylether/methanol/water/90% formic acid (95/4/1/1), and bezene/methanol/acetic acid (24/2/1), with R(f) values (min-max) of 0.09-0.18, 0.88-0.95, 0.06-0.15, 0.39-0.47 and 0.05-0.12, respectively. The recovered metabolite from the TLC plate displayed UV absorption peaks at 212, 244, 250, 256 and 261 nm. The proposed formula of the main component of the metabolite was C16H12N3O6. The TLC-purified metabolite induced symptom of discoloration on detached pea leaves.     

The life history parameters of Diaphanosoma excisum (Cladocera), grown in different culturing media

Life history parameters of Diaphanosoma excisum (SARS) were derived from culture tube experiments with media comprising mohua oil cake, mustard oil cake, cow dung and rice bran. Body length, longevity, fecundity and progeny production were highest in rice bran medium. First reproduction time (A) in different media ranged from 2 to 6 days, while the reproduction peak (T) varied from 4 to 13 days. The total offspring per female (S) was directly dependent upon the mean number of eggs per female per life span. The intrinsic (r) and finite rate (e^r) of natural increase were larger for rice bran than for other media.     

Pine wilt disease as promising causal agent of the mass mortality of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima in the field

The causal agent of the mass mortality of field populations of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima (PAA) was investigated with special respect to the involvement of pine wilt disease. Wood chips, branches and/or increment cores for detecting the pinewood nematode, feeding marks of the vector insect and environmental stress in the past, respectively, were taken from live and dead PAA trees grown in three locations, Yaku-shima and Tanega-shima Islands and a plantation in Kagoshima City, from 1997 to 1998. Five trees died after the spring of 1996 and, of these, four were inhabited by the pinewood nematode. Feeding marks of the vector insects were found on the branches of all dead trees and most of the live trees investigated. These results suggest that the infection of pine wilt disease in PAA trees occurs in the field. Annual ring growth of the sample trees showed neither intervention nor growth reduction, which implies strong environmental stress that may cause mortality in PAA trees.     

Improved culture media for growth of Bradyrhizobium japonicum

A freshly-prepared yeast extract at 30 or 50 g/l improved the growth of Bradyrhizobium japonicum SEMIA 587 in a 5-l stirred fermenter. Monosodium glutamate or a commercial yeast extract at 2.0 g/l almost doubled cell mass productivity and cell viability when added at the end of the first exponential growth phase.The authors are with the Divisão de Quimica, Agrupamento de Biotecnologia, Instituto de Pesquisas Tecnológicas do Estado de São Paulo, S/A.-IPT-Cidade Universitária s/n., Caixa Postal 7141, CEP 01064-970, São Paulo, SP, Brazil     

Screen of safflower (Carthamus tinctorius L.) genotypes against Phytophthora drechsleri and Fusarium solani,the causal agents of root rot disease

Safflower (Carthamus tinctorius L.) plants were affected by a severe root rot disease caused by Phytophthora drechsleri and Fusarium solani in Isfahan province of Iran during 2005–2007. Disease incidence was more than 30% in severely infected fields. Twenty-one safflower genotypes, including six local cultivars and 15 internal pure lines were evaluated for their resistance to root rot disease in laboratory and greenhouse conditions. Safflower seedlings were evaluated for lesion length on infected roots in laboratory, as well percentage of live seedlings in greenhouse. The results indicated a high negative correlation between lesion length on roots and percentage of live seedlings. The most resistant and susceptible genotypes to P. drechsleri were identified as pure line Karaj row 12 (KW12) and cultivar Koseh with lesion lengths of 10.01 and 15.51?mm on roots and 45.60 and 18.00% live seedlings, respectively. The most resistant genotype to F. solani was identified as pure line KW11 with a lesion length of 9.31?mm on roots as well 62.80% live seedlings. The most susceptible genotypes were identified as cultivar Koseh and pure lines KW2 and KW3 with lesion lengths of 13.29, 12.72 and 12.13?mm on roots and 25.60, 28.40 and 28.40% live seedlings, respectively. The most resistant genotypes to both P. drechsleri and F. solani were identified as pure lines KW15 and KW9 with a 55.40% live seedlings. The most susceptible genotypes were cultivars Koseh, Goldasht and pure lines KW6, KW3 and KW2 with 35.40, 35.40, 35.40, 37.60 and 37.60% live seedlings in greenhouse, respectively.     

The endobiotic thallus ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The endobiotic thallus ofPhysoderma maydis is characterized by the presence of an extremely fine rhizomycelium which passes through the host cell wall, allowing the spread of the disease, and irregularly shaped turbinate cells, which may be septate or nonseptate and which are in close association with developing resting sporangia. The formation of the resting sporangium wall is first seen as localized depositions on the rounded surface of the sporangium and only later on the flattened surface of the sporangium which will form the operculum. The substructure of the resting sporangium wall is typical for members of theBlastocladiales. The resting sporangium is contiguous with the rhizomycelium during development and is finally sealed-off from the rhizomycelium by a further deposition of wall material. After the sealing-off of the resting sporangium from the rhizomycelium the content of the sporangium is compartmentalized and the two inner wall layers are deposited. The centre of the sporangium is filled with an electron dense accretion. At the periphery of the sporangium is a layer of lipid bodies. Between the lipid bodies and the central electron dense accretion is a thin layer of cytoplasm which contains the nuclei. The outer surface of the resting sporangium is smooth.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

Effect of metabolites ofAspergillus niger andTrichoderma viride on development and structure of radicle of cocoa (Theobroma cacao) seedlings

Both the volume and concentration of filtrate ofAspergillus niger andTrichoderma viride influenced the development of the radicles of cocoa seedlings. Radicles placed in Petri dishes containing filter paper moistened with 20 and 30 ml of undiluted filtrate and filtrate dilutions of 1∶1 and 1∶2 failed to develop lateral roots and eventually died. The culture filtrate ofA. niger was more repressive. Radicles in the same volume of filtrate (20 and 30 ml) of higher dilutions (1∶5 and 1∶10) developed lateral roots and survived. Radicles placed in less volume (≤10 ml) survived and produced lateral roots irrespective of concentration of filtrates. Development of the radicles and roots in the control was consistently better (P=0.05) than in filtrate solutions of eitherA. niger orT. viride. The hypocotyls of seedlings under the influence of metabolites ofA niger showed greater cambial activity and formed xylem vessels and tracheids with larger lumina.