Insecticidal and fungicidal activity of new synthesized chitosan derivatives

Chitosan, the N-deacetylated derivative of chitin, is a potential biopolysaccharide owing to its specific structure and properties. In this paper, we report on the synthesis of 24 new chitosan derivatives, N-alkyl chitosans (NAC) and N-benzyl chitosans (NBC), that are soluble in dilute aqueous acetic acid. The different derivatives were synthesized by reductive amination and analyzed by 1H NMR spectroscopy. A high degree of substitution (DS) was obtained with N-(butyl)chitosan (DS 0.36) at a 1:1 mole ratio for NAC derivatives and N-(2,4-dichlorobenzyl)chitosan (DS 0.52) for NBC derivatives. Their insecticidal and fungicidal activities were tested against larvae of the cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), the grey mould Botrytis cinerea Pers (Leotiales: Sclerotiniaceae) and the rice leaf blast Pyricularia grisea Cavara (Teleomorph: Magnaporthe grisea (Hebert) Barr). The oral feeding bioassay indicated that all the derivatives had significant insecticidal activity at 5 g kg(-1) in artificial diet. The most active was N-(2-chloro-6-fluorobenzyl)chitosan, which caused 100% mortality at 0.625 g kg(-1), with an estimated LC50 of 0.32 g kg(-1). Treated larvae ceased feeding after 2-3 days; the mechanism of action remains unknown. In a radial hyphal growth bioassay with both plant pathogens, all derivatives showed a higher fungicidal action than chitosan. N-Dodecylchitosan, N-(p-isopropylbenzyl)chitosan and N-(2,6-dichlorobenzyl)chitosan were the most active against B cinerea, with EC50 values of 0.57, 0.57 and 0.52 g litre(-1), respectively. Against P grisea, N-(m-nitrobenzyl)chitosan was the most active, with 77% inhibition at 5 g litre(-1). The effect of different substitutions is discussed in relation to insecticidal and fungicidal activity.     

Biological control ofBotrytis cinerea in residues and flowers of Rose (Rosa hybrida)

Microbial isolates from living petals, petal residues and leaf residues of rose, and from laboratory collections, were evaluated for control ofBotrytis cinerea in rose. In leaf residues artificially infested withB. cinerea, isolates of the filamentous fungiGliocladium roseum, FR136 (unidentified) andTrichoderma inhamatum reduced sporulation of the pathogen by >90%, other filamentous fungi were 25–90% effective, and those of yeasts and bacteria were <50% effective. In artificially inoculated petal residues, no microbe reduced sporulation ofB. cinerea by >75%, but isolates ofCladosporium oxysporum and four yeasts were 51–75% effective, and three filamentous fungi, eight yeasts andBacillus subtilis isolates were 26–50% effective. Isolates ofT. inhamatum, C. oxysporum andG. roseum performed best againstB. cinerea among isolates evaluated in leaf residues naturally infested with the pathogen and indigenous microorganisms. Totals of ten isolates of filamentous fungi (includingC. oxysporum andC. cladosporioides), two of yeasts and five ofBacillus subtilis completely prevented lesion production byB. cinerea in detached petals, and a further six isolates of filamentous fungi (includingG. roseum) and six yeasts were 90–99% effective. Isolates ofC. oxysporum, C. cladosporioides andB. subtilis, the most effective microorganisms againstB. cinerea in flower buds, reduced number of lesions in the range of 42–65% compared with 59–89% for à standard fungicide (vinclozolin). It is suggested that application of leading antagonists Jo living rose leaves and flowers should optimize control of inoculum production byB. cinerea when the tissues die. Optimal biocontrol of lesion production in flower buds requires a better understanding of the microenvironment of petals.     

Mode of action of the anilino-pyrimidine fungicide pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea

The effect of pyrimethanil on the levels of cell wall degrading enzymes secreted by Botrytis cinerea Pers. was investigated in diseased plant tissues and in liquid B. cinerea cultures. Total proteinase activity isolated from infected carrot slices which were treated with 5.0 μM pyrimethanil was decreased by 76%, 3 d after inoculation. Polygalacturonase, cellulase, proteinase and laccase activities were all decreased in the medium of three day-old cultures grown in the presence of pyrimethanil. The pyrimethanil concentrations resulting in 50% reduction in total enzyme activities (IC₅₀) were approximately 0.25 μM for polygalacturonase, cellulase and proteinase, and approximately 1.0 μM for laccase. No significant growth inhibition was observed at these pyrimethanil concentrations. Pyrimethanil did not inhibit the enzymes directly, nor did it inhibit the synthesis of cytosolic proteins. Therefore, it was proposed that the fungicide inhibits protein secretion at a post-translational stage in the secretory pathway. Large differences were found in the effects of pyrimethanil on the growth of B. cinerea in liquid cultures and on agar plates, depending on the composition of the medium. In liquid media containing cellulose and protein as carbon and nitrogen sources, growth inhibition occurred at 5.0 μM pyrimethanil, whilst no growth inhibition was observed with 50 μM pyrimethanil in malt extract. Similarly, growth occurred on potato/dextrose agar (PDA) at 0.5 μM pyrimethanil, but no growth was seen at this concentration on agars containing cellulose and protein. Thus it appears that pyrimethanil is most active in media where the fungus has to utilise extracellular enzymes to mobilise the nutrients it requires for growth.     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

Biological control of botrytis leaf blight of onion byGliocladium roseum applied as sprays and with fabric applicators

Gliocladium roseum (5×10⁶ conidia ml^–1) and chlorothalonil (Bravo 500) were compared in two field tests for effectiveness in suppressing leaf blight caused byBotrytis squamosa in cooking onions. The biological control agent and fungicide were applied with sprayers and with specially designed fabric applicators that were mounted interchangeably on an aluminium carriage with bicycle wheels. In the applicators, inoculum and fungicide gravitated from a reservoir down curtains of denim strips and onto portions of onion leaves contacted by the strips. Initial applications were timed using a leaf blight forecasting system (BOTCAST) and three or four subsequent applications were made at weekly intervals. When compared to water checks,G. roseum applied as sprays or by applicators reduced density of leaf spots by about 50–58% during middle and late stages of epidemics. The antagonist was about half as effective as chlorothalonil in suppressing density of spots, regardless of application method. The applicators delivered inoculum in similar densities to proximal and distal halves of leaves, and used at least 35% less volume of inoculum compared to the sprayers. Density of inoculum on leaves treated by applicators was about the same as in spray-treated leaves when canopies were sparse, but less when canopies were dense. It is concluded thatG. roseum has good potential for controlling leaf blight sufficiently to avoid economic yield losses.     

草莓灰霉病菌的培养、毒素提取及生物测定

 草莓灰霉病菌(Botrytis cinerea Persoon)在25℃、pH3~4的Peberdy培养基中生长良好,并可产生引起草莓愈伤组织细胞死亡的毒素。产生毒素的高峰期为静置培养20天。灰霉菌发酵液中的毒素可用氯仿进行提取,经提取后的毒素液在269nm处有吸收峰。本研究建立了利用草莓愈伤组织细胞的荧光活性测定毒力的方法。     

咯菌腈对草莓灰霉病Botrytis cinerea的毒力及防效研究初报

用生长速率法测定了咯菌腈、多菌灵、硫菌·霉威、苯醚甲环唑4种药剂对草莓灰霉病Botrytis cinerea的毒力。结果表明,4种药剂对草莓灰霉菌菌丝的生长都有不同程度的抑制作用,随药剂浓度增加抑制作用增强,其EC50值分别为0.0472、95.7193、0.4854、1.3611 mg/L,其中咯菌腈的毒力最强。田间防治试验结果表明:2.5%咯菌腈FC每667 m2施药量120 mL时防效为83.7%,80 mL时防效为67.7%;65%硫菌·霉威WP每667m2施药量100 g时防效为65.3%。     

An inhibitor of polyamine biosynthesis — Difluoromethylornithine — and the polyamine spermidine for the control of gray mold(Botrytis Cinerea)

Difluoromethylornithine (DFMO) — an inhibitor of polyamine biosynthesis, and the polyamine spermidine (Spd) reduced gray mold of tomato, pepper, eggplant, bean andSenecio sp. leaves, and of rose petals by 37–88% when applied at 0.1–1.0 mM each. Higher doses did not result in better control. The disease was also reduced significantly on tomato fruits by 1.0 inM DFMO and by 0.1–1.0 mM Spd, and on cucumber fruits by 0.1–1.0 mM of both compounds, but not on grape berries. The combination of 0.2 mM DFMO with 1.0 mM Spd controlled gray mold ofSenecio sp. and tomato leaves additively better than either treatment alone, whereas this effect was not observed in leaves of lettuce and pepper. Ethylene production was reduced significantly by Spd applied to leaves of tomato and pepper, but not by DFMO. Linear growth and germination of the fungus were affected by lower concentrations of DFMO (ED50 0.12–0.97 and 1.4, respectively) as compared with Spd. Spermidine and DFMO controlled white mold(Sclerotinia sclerotiorum) as effectively as did the fungicide benomyl. Contribution from the Agricultural Research Organization. No. 3195-E, 1991 series.     

Calcium reducesBotrytis c1nerea damages to plants ofRuscus hypoglossum

Plants ofRuscus hypoglossum L. were grown in containers with perlite, an inert medium, or with soil, under similar-to-commercial conditions, for three winter seasons. Constant fertilization with soluble calcium [Ca(NO₃)₂] through the root zone resulted in a reduction of up to 78% in postharvest rot caused by artificial inoculation withBotrytis cinerea Pers. ex Fr. Control plants were treated by fertilization with N,P,K and all other elements, identical to the fertilizer containing calcium. Foliar application of Ca(NO₃)₂ to ruscus bushes or application of this compound or of CaCl₂ or CaSO₄ to mature cut branches standing in water, resulted also in a significant reduction in postharvest phylloclade rot. Application of CaSO₄ and Ca(H2PO₄)₂ + CaSO₄ to ruscus plants, from fertilizers containing 22% Ca and releasing the cation gradually, effectively reduced the susceptibility of cut branches to infection by_{B. cinerea.}