Arbuscular mycorrhizal fungi influence water relations, gas exchange, abscisic acid and growth of micropropagated chile ancho pepper (Capsicum annuum) plantlets during acclimatization and post-acclimatization

Little is known about the role of arbuscular mycorrhiza fungi (AMF) on physiological changes of micropropagated plantlets during acclimatization and post-acclimatization. Using chile ancho pepper (Capsicum annuum L. cv. San Luis), measurements were made of water relations, gas exchange, abscisic acid (ABA), plantlet growth and AMF development. Plantlets had low photosynthetic rates (A) and poor initial growth during acclimatization. Relative water content (RWC) decreased during the first days after transfer from tissue culture containers to ex vitro conditions. Consequently, transpiration rates (E) and stomatal conductance (gs) declined, confirming that in vitro formed stomata were functional and able to respond ex vitro to partial desiccation--thus avoiding excessive leaf dehydration and plant death. Colonization by AMF occurred within 3 days after inoculation. Colonized plantlets had lower leaf ABA and higher RWC than noncolonized (NonAMF) plantlets during peak plant dehydration (6 days after plant transfer)--and a higher A and gs as early as days 5 and 7. During post-acclimatization [after day 8, when RWC increased and stabilized], A increased in all plantlets; however, more dramatic changes occurred with AMF plantlets. Within 48 days, 45% of the roots sampled of inoculated plantlets were colonized and had extensive arbuscule development. At this time, AMF plantlets also had greater E, A, leaf chlorophyll, leaf elemental N, P and K, leaf dry biomass and leaf area, fruit production and differences in carbon partitioning [lower root/shoot ratio and higher leaf area ratio] compared with NonAMF plantlets. Rapid AMF colonization enhanced physiological adjustments, which helped plantlets recover rapidly during acclimatization and obtain greater growth during post-acclimatization.     

Arbuscular mycorrhizal fungi enhance fruit growth and quality of chile ancho (Capsicum annuum L. cv San Luis) plants exposed to drought

The effect of arbuscular mycorrhizal fungi (AMF) and drought on fruit quality was evaluated in chile ancho (Capsicum annuum L. cv San Luis). AMF treatments were (1) Glomus fasciculatum (AMFG), (2) a fungal species consortium from the forest “Los Tuxtla” in Mexico (AMFT), (3) a fungal species consortium from the Sonorian desert in Mexico (AMFD), and (4) a noninoculated control (NAMF). Plants were exposed to a 26-day drought cycle. Fruit quality was determined by measuring size (length, width, and pedicel length), color, chlorophyll, and carotenoid concentration. Under nondrought conditions, AMFG produced fruits that were 13% wider and 15% longer than the NAMF treatment. Under nondrought conditions, fruit fresh weight was 25% greater in the AMFG treatment compared to the NAMF. Under drought, fruits in the AMFT and AMFD treatments showed fresh weights similar to those in the NAMF treatment not subjected to drought. Fruits of the AMFG treatment subjected to drought showed the same color intensity and chlorophyll content as those of the nondroughted NAMF treatment and carotenoid content increased 1.4 times compared to that in the NAMF not exposed to drought. It is interesting to note that fruits in the AMFD treatment subjected to drought and the NAMF treatment not exposed to drought reached the same size. AMFD treatment increased the concentration of carotenes (1.4 times) under nondrought conditions and the concentration of xanthophylls (1.5 times) under drought when compared to the nondroughted NAMF treatment.     

Photoinhibition during acclimatization of micropropagatedSpathiphyllum “Petite” plantlets

Summary MicropropagatedSpathiphyllum “Petite” plantlets were acclimatized at low- or high-light intensities [photosynthetic photon flux density (PPFD) 100 or 300 μmol·m⁻²·s⁻¹]. During the first days chlorophyll fluorescence measurements show a partial photoinhibition of the photosynthetic apparatus, expressed by a decrease of the variable over maximal fluorescence ratio (F_v/F_m). This inhibition of F_v/F_m was significantly higher for plants grown at high-light intensity, leading to a photooxidation of chlorophyll. Newly formed leaves were better adapted to the ex vitro climatic condition (as shown by the increase of the F_v/F_m ratio) and had a higher net photosynthesis compared with in vitro formed leaves. Nevertheless, plants grown at 300 μmol·m⁻²·s⁻¹ were photoinhibited, compared with those at 100 μmol·m⁻²·s⁻¹. A sudden exposure to high-light intensity of 1-, 10- or 25-d-old transplanted plants (shift in PPFD from 100 to 300 μmol·m⁻²·s⁻¹) gave a linear decrease of F_v/F_m over a 12-h period, which was reflected in a 50% reduction of net photosynthesis. No significant interaction between day and hour was found, indicating high-light exposure causes the same photoinhibitory effect on in vitro and ex vitro formed leaves.     

Humid incubation period and plantlet age influence acclimatization and establishment of micropropagated grapes

Summary In vitro rooted grape (Vitis vinifera L.) plantlets (4 or 8 wk from culturing microcuttings) were plantedex vitro in polythene sachets (24×12 cm) filled to one-third their height with planting mixture. The sachets were misted, closed, and incubated at ambient temperature (25–30°C) under 16 h photoperiod (40–50 μ E·m^−2.·sec⁻¹) for 1,2, or 3 wk before opening. Maximum establishment with no or minimum damage toin vitro formed leaves was obtained with 3 wk of closed sachet incubation in both age groups. Opening the sachet at 2 wk from planting resulted in marginal scorching of lower leaves and some reduction in establishment and vigor. Opening at 1 wk led to severe leaf scorching and significant reduction in establisment particularly in 4-wk-oldin vitro plantlets while growth was more affected in 8-wk-old ones. Relative humidity ofin vitro culture vessel was 68–75% while closed sachets had RH values of 45–77% depending on length of incubation and ambient RH. Diurnal variation in RH of sachet in relation to ambient RH was the major factor that facilitated acclimatization rather than the overall fall in RH during the period of closed incubation. Satisfactory acclimatization of plantlets to withstand the open sachet RH (50–55%) by 3 wk and the ambient RH (30–40%) by 4 wk was achieved. Monitoring water loss from detached leaves of plantlets showed a significant reduction between the date of planting and Week 3, and again between Weeks 3 and 4. Comparison of growthin vitro andex vitro suggested that shifting toex vitro earlier was more beneficial. This observation was confirmed by transferring 3-, 4-, and 5-wk-old plantlets fromin vitro rooting medium toex vitro and recording the growth at 8 wk fromin vitro culturing when 3 wkin vitro plus 5 wkex vitro combination showed maximum vigor. The leftover stumps after subculturing of 1–4-mo.-old stock cultures could also be effectively used forex vitro establishment.     

Water stress and photoinhibition in acclimatization ofRosa hybrida plantlets

Summary MicropropagatedRosa hybrida plantlets were simultaneously rooted and acclimatized under 100 and 200 μmol m⁻² s⁻¹ light for 2 wk. At the end of the first week of acclimatization, the plantlets were transferred onto a low water potential medium (from −0.06 MPa to −0.3 MPa). Dry weight was decreased by increased hight and low water potential. Photoinhibition of photosynthesis, expressed as a decrease in Fv/Fm ratio and ΦPSII and an increase in 1 −qp, occurred in plants grown under 200 μmol m⁻² s⁻¹. When high light (200 μmol m⁻² s⁻¹) and water stress were applied simultaneously, their effects on chlorophyll fluorescence parameters depended on stress duration; after 1 d of water stress, photoinhibition was more pronounced; after 7 d of stress, Fv/Fm ratio and ΦPSII were higher than after 1 d of stress; photoinhibition was reduced. This suggests that after a 1-d stress, the effect of water stress alone included a superimposed effect of photoinhibition to which the water-stressed plants were sensitized; after 7 d, plantlets had adapted to water stress. The photoprotective effects under high light might result in energy dissipative mechanisms linked to photochemical and nonphotochemical quenching other than CO₂ fixation.     

Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

Origin of plantlets and callus obtained from chile pepper anther cultures

Summary Androgenesis occurred from chile pepper (Capsicum annuum L.) anthers incubated in a continuous warm environment (29° C) with continuous light. Forty plantes and embryoids were retrieved from anther cultures and anllyzed for isozyme markers. Of these, 35 exhibited a single allele for markers suggesting microspore origin, while 5 were heterozygous indicating somatic tissue origin. Chromosome numbers were confirmed for 21 plantlets, of which 16 were haploid and 5 were diploid. However, two plants exhibited a single allele for an isozyme marker but possessed the diploid chromosome number, suggesting spontaneous doubling. Anther cultures also produced callus. Nearly 92% of the slow-growing calli sampled were heterozygous for the isozyme marker, suggesting somatic tissue origin. More than 46% of the fast-growing calli exhibited only one allele for the marker, indicating microspore origin. Callus did not regenerate plantlets. The occurrence of both heterozygous and homozygous diploid plantlets from pepper anther cultures has important implications for applied breeding programs.     

Improvement of acclimatization of micropropagated grapevine: Photosynthetic competence and carbon allocation

Grapevine plantlets multiplied in vitro were acclimatized at 40 or 90 μmol m⁻² s⁻¹ photon flux density for 12 or 16 h per day, respectively. In the high-light regime a decrease in total chlorophyll and an increase in chlorophyll a/chlorophyll b ratio occurred. However, at high-light intensity lower photosynthetic capacities and higher apparent photosynthesis were measured than at the low-light regime. In leaves expanded during acclimatization, the light compensation point was higher in plantlets under high-light while quantum yield was higher in low-light conditions. High-light also gave rise to an increase in carbohydrate concentration. As a whole, the results suggest that high-light increases carbon assimilation and growth although with a low investment in the photosynthetic apparatus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.     

Improvement of isolated microspore culture of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.     

In vitro rooting of sentang shoots (Azadirachta excelsa L.) and acclimatization of the plantlets

Summary In vitro proliferated sentang shoots were cultured onto half-strength Murashige and Skoog (MS) medium containing combinations of 1-naphthyleneacetic acid (NAA) and indole-3-butyric acid (IBA). Sentang shoots were unable to root in the absence of both auxins. A combination of 0.5 mg NAA per and 1 mg IBA per 1 induced the most shoots to form roots. With the addition of 2.5 g activated charcoal per 1 into half-strength MS medium containing 0.5 mg NAA per 1 and 1 mg IBA per 1, roots were more numerous and longer. Substances like gelrite and phloroglucinol and sugar content which would commonly influence in vitro rooting were inhibitory to adventitious root formation of sentang. Maximal rooting of 100% was achieved in “Culture Pack,” made of fluorocarbon polymer film containing charcoal-free medium with 0.5 mg NAA per 1 and 1 mg IBA per 1. Rooted shoots were acclimatized for 4 wk. Overall survival was 80%. These findings suggest the use of Culture Pack as the culture vessels, with 0.5 mg NAA per 1 and 1 mg IBA per 1 in half-stength MS media to effectively induce roots in sentang shoots.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

Elongation, rooting and acclimatization of micropropagated shoots from mature material of hybrid larch

Factors were defined for elongation, rooting and acclimatization of micropropagated shoots ofLarix x eurolepis Henry initiated from short shoot buds of plagiotropic stecklings serially propagated for 9 years from an 8-year-old tree. Initiation and multiplication were on Schenk and Hildebrandt (SH) medium supplemented with 5 M 6-benzyladenine (BA) and 1 M indole-butyric acid (IBA). Stem elongation was obtained in 36% of the shoots on SH medium containing 0.5 M BA and 63% of the remaining non-elongated shoots initiated stem elongation after transfer on SH medium devoid of growth regulators. Rooting involved 2 steps: root induction on Campbell and Durzan mineral salts and Murashige and Skoog organic elements, both half-strength (CD-MS/2), supplemented with 1 M of both naphthaleneacetic acid (NAA) and IBA, and root elongation following transfer to CD-MS/2 medium devoid of growth regulators. Repeating this 2-step sequence yielded up to 67% rooted shoots. Acclimatization of plantlets ranged from 83% to 100%. Over 300 plants were transferred to the greenhouse; some showed plagiotropic growth.     

Both common and specific genetic factors are involved in polygenic resistance of pepper to several potyviruses

Absolute resistance to potato virus Y pathotype 0 (PVY 0), potyvirus E and chili veinal mottle virus (CVMV) and a partial resistance to potato virus Y pathotype 1,2 (PVY 1,2) were found in an Indian pepper line, Perennial. In the doubled haploid (DH) progeny from the F₁ of a cross Perennial by Yolo Wonder, resistance to CVMV was confered by two independent genes, one with a clear dominant effect. Resistance to PVY and potyvirus E was quantitatively expressed and controlled by several recessive genetic factors. Genetic analysis showed that fewer resistance factors were necessary to explain resistance to PVY (0) and potyvirus E than resistance to PVY(1,2). Genetic correlations between resistances to the different potyviruses in the DH progeny showed that most of genetic factors involved in PVY(0) resistance appear to be also involved in potyvirus E resistance, and some of these polyvalent factors may be also involved in PVY(1,2) resistance but, in this case, additional specific genes were necessary. One of the two CVMV resistance genes seems to be implicated in potyvirus E resistance. Thus, the polygenic resistance of Perennial to these potyviruses was due both to polyvalent genetic factors, i.e. factors that apparently interact with several viruses, and strain-specific genetic factors.     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.     

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 mol m-² s-^-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation ( 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L^–1 for 3 h and then subculturing on MS medium without growth regulators.     

Physiological and growth changes in micropropagated Citrus macrophylla explants due to salinity

Salinity is one of the major abiotic stresses affecting arable crops worldwide, and is the most stringent factor limiting plant distribution and productivity. In the present study, the possible use of in vitro culture to evaluate the growth and physiological responses to salt-induced stress in cultivated explants of Citrus macrophylla was analyzed. For this purpose, micropropagated adult explants were grown in proliferation and rooting media supplemented with different concentrations of NaCl. All growth parameters were decreased significantly by these NaCl treatments; this was accompanied by visible symptoms of salt injury in the proliferated shoots from 60 mM NaCl and in the rooted shoots from 40 mM NaCl. Malondialdehyde (MDA) increased with increasing salinity in proliferated shoots, indicating a rising degree of membrane damage. The concentration of total chlorophyll significantly decreased in the presence of NaCl, and this effect was more pronounced in the rooted explants. The Na⁺ and Cl⁻ concentrations in the explants increased significantly with the salinity level, but Cl⁻ levels were higher in the proliferated explants than in the rooted explants. For osmotic adjustment, high concentrations of compatible solutes (proline and quaternary ammonium compounds—QAC) accumulated in salt-stressed plants in proliferation, but differences were not observed in rooted explants. In proliferation, proline and QAC were highly correlated with the sodium and chloride concentrations in the explants, indicating a possible role of these compounds in osmotic adjustment. The plant concentrations of NO₃⁻, K⁺, Mg²⁺, Ca⁺ and Fe were also affected by the NaCl concentration of the medium. We suggest that the important deleterious effects in the in vitro explants of Citrus macrophylla grown at increasing NaCl concentrations were due mainly to toxic effects of saline ions, particularly Cl⁻, at the cellular level.     

Combined effect of salinity and excess boron on plant growth and yield

Plants are likely to be affected by simultaneous salinity and boron (B) toxicity stresses due to exposure to soils with high levels of naturally occurring salinity and B, or due to irrigation with water containing high levels of salts, including B. Inadequate information regarding the response of plants to the combination of excess B and salinity on plant growth and yield is available, and there is no consensus concerning mutual relations between salinity stress and B toxicity. Growth and yield of bell pepper (Capsicum annuum L.) were measured at different B and salinity levels in two greenhouse experiments. The results from these experiments and from published data for wheat, tomato and chickpea were analyzed according to the Abbott method to define the combined effect of B and salinity on plant growth and yield. Application of the Abbott method for the experiments on peppers generally implied an antagonistic relationship for excess B and salinity. In other words, toxic effects on growth and yield were less severe for combined B toxicity and salinity than what would be expected if effects of the individual factors were additive. Similar antagonistic characteristics were found using data from three of the five studies reported in the literature. The mechanism of relationships between B and salinity in plants is not clear and several options are discussed. Prominent among the possible explanations are reduced uptake of B in the presence of Cl and reduced uptake of Cl in the presence of B.     

Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici

This study investigated the expression pattern of genes encoding for a basic PR-1 protein, a basic beta-1,3-glucanase, a peroxidase, and a sesquiterpene cyclase involved in defense responses in three pepper cultivars with different levels of resistance to Phytophthora capsici. All genes were up-regulated in infected stems of the pepper cultivars, with expression being detected 8h post-inoculation. mRNA levels of these genes increased markedly by 24h post-inoculation, and maximal induction levels were observed for the PR-1 and sesquiterpene cyclase genes. PR-1, peroxidase, and sesquiterpene genes were always expressed at higher levels in resistant cultivars than in the susceptible cultivar, although up-regulation was observed in both, suggesting that the differences between these pepper genotypes in susceptibility and resistance are a matter of the timing and magnitude of the defense response.