Growth of Xanthomonas campestris pv. campestris populations at constant and variable temperatures

Quantitative data were collected to describe the relation between temperature and growth of the cabbage black rot pathogen,Xanthomonas campestris pv.campestris (Xcc). Relative growth rates derived from experiments at constant temperatures were used in dynamic simulation of bacterial population development. The relative growth rates were adequate to simulate growth ofXcc populations at constant temperatures but overestimated growth of populations at variable temperatures. This finding gives rise to the hypothesis, that under field conditions, disease development is slower than is expected on the basis of growth parameters obtained from studies with constant temperatures.     

Comparative biology and life tables of <Emphasis Type="BoldItalic">Trichogramma aurosum</Emphasis> on <Emphasis Type="BoldItalic">Cydia pomonella</Emphasis> at constant temperatures

The influence of constant temperatures on biological parameters of German strains of Trichogramma aurosum Sugonjaev & Sorokina (Hymenoptera: Trichogrammatidae) was evaluated in the laboratory on eggs of Cydia pomonella L. (Lepidoptera: Tortricidae). Development time and longevity of all strains were decreased as temperature increased. Development time of the strains differed significantly only when exposed to 15°, 20°, and 25°C. Cumulative fertility and longevity differed significantly at 15° and 20°C. Realized fertility differed significantly at all constant temperatures. Emergence rates of all strains were less than 65% and were decreased even further as temperature increased. Female-biased sex ratio ranged from 65% to 100% at all constant temperatures. The low temperature threshold for T. aurosum was 10°C and the mean number of degree-days at 15°, 20°, 25° and 30°C was 175, 183, 173 and 185, respectively. The Bavarian strain tolerated high temperatures and had the highest parasitization capability, while the Hessian strain had the lowest parasitization at all temperatures. Fertility life table analysis revealed a major effect of temperature on the population growth parameters. Net reproductive rate was highest at intermediate constant temperatures in all strains, with the highest rate recorded for the Bavarian strains at all constant temperatures. Mean cohort generation time, and population doubling time decreased as temperature increased. The daily intrinsic rate of increase and finite rate of increase were positively correlated with temperature. The relevance of our results is discussed in the context of climatic adaptation, intraspecific variability and biological control.     

Germination behaviour in seeds of Diplotaxis erucoides and D. virgata

The seed germination behaviour of four populations each of Diplotaxis erucoides (L.) DC. and D. virgata (Cav.) DC, two weedy Brassicaceae species widely distributed in the Mediterranean region, was studied under controlled light, temperature and moisture conditions. Germination rates in D. virgata were generally higher at alternating (25/15^C) and low (15^C) temperatures, whereas in D. erucoides optimal germination rates were achieved at higher temperatures (25^C). No correlation between seed weight and germination was found in either species. In D. virgata, the geographic origin of the seed had an important effect on germination percentage. Great germination variability was found among the populations and even among seeds belonging to the same population. In this species, the application of gibberellic acid stimulated germination, especially in the populations with higher dormancy levels. Dormancy was found in mature D. virgata seeds but not in those of D. erucoides. In both species, germination percentages decreased after a certain period of storage at low temperatures. This period varied depending on the species and the population considered.     

A hydrothermal seedling emergence model for Conyza bonariensis

Conyza bonariensis is a South American native annual Asteraceae that has been introduced to the Mediterranean, where it behaves as a ruderal plant and a weed that is difficult to control in several crops. The development of predictive models can contribute to control measures at early growth stages, but currently there are no studies to predict seedling emergence of Conyza species. Our objectives were to develop and evaluate a model for predicting emergence response of C. bonariensis to the soil hydrothermal environment. A hydrothermal seed germination model was fitted to time course germination data from germination tests carried out at different constant temperatures and water potentials with the aim of establishing the hydrothermal parameters characterising C. bonariensis seed germination. The relationship between cumulative seedling emergence and cumulative hydrothermal time under field conditions was analysed using the Gompertz function. Model development was based on 2 years' data from a field experiment. Base temperature and base water potential for seed germination were estimated at 10.6°C and ?0.70 ± 0.151 MPa, respectively. The emergence model showed a very good fit to the experimental data. According to this model, seedling emergence starts at 15 accumulated hydrothermal time (HTT) after sowing, and 50 and 95% emergence is completed at 53 HTT and 105 HTT, respectively. For model evaluation, independent field experiments were carried out in two localities. Cumulative seedling emergence was accurately predicted by the model. Results indicate that this model can be useful as a predictive tool contributing to effective control of C. bonariensis populations.     

The effect of a mixture of seed-borne Microdochium nivale var. majus and Microdochium nivale var. nivale infection on Fusarium seedling blight severity and subsequent stem colonisation and growth of winter wheat in pot experiments

Greenhouse experiments were conducted in order to determine the impact of seed-borne Microdochium nivale var. nivale and var. majus inoculum, and seed treatment with a carboxin+thiram mixture, on the development of seedling blight, and on subsequent stem colonisation and growth of winter wheat (cv. Cadenza). Experiments were conducted at temperatures favourable (3°C) and unfavourable (22°C) to M. nivale. Seed-borne inoculum resulted in seedling blight symptom development when plants were grown at 3°C, but not when plants were grown at 22°C. For seedlings grown at 3°C, plants arising from heavily blighted seedlings developed more severe symptoms of stem colonisation, when compared with those arising from seedlings from carboxin+thiram treated seeds. In addition, the vigour of such plants (assessed by determining the number of tillers and ears per plant, stem length, green leaf area, dry weight and yield) was also significantly lower than for plants arising from carboxin+thiram treated seeds. Microdochium nivale var. majus and var. nivale appeared to have little effect on plant vigour from seedlings grown at 22°C. This is the first recorded incidence of seedling blight affecting subsequent plant growth. Microdochium nivale var. majus and var. nivale stem colonisation increased from growth stage (GS) 40–49 to harvest in plants raised from seedlings grown at both temperatures. Microdochium nivale var. majus and var. nivale were isolated from the second node at GS 40–49 and the third node at harvest of plants from seedlings grown at 3°C. For plants from seedlings raised at 22°C, M. nivale var. majus and var. nivale were isolated from the first node at GS 40–49 and the second node at harvest. Carboxin+thiram seed treatment decreased the extent and severity of stem colonisation on plants from seedlings grown at 22°C.     

Artificial infection of Vaccinium vitis-idaea L. and defence responses to Exobasidium species

An inoculation method for Exobasidium splendidum and Exobasidium vaccinii was developed on the dwarf shrub Vaccinium vitis-idaea. Using inoculated ramets, we investigated whether there are differences between V. vitis-idaea populations in the susceptibility to Exobasidium infections and whether the defence reaction of V. vitis-idaea is visible at a molecular level. Sixteen V. vitis-idaea clones from four populations were propagated in tissue cultures and the ramets were inoculated with E. splendidum or E. vaccinii fungi. The expression of three flavonoid biosynthetic genes (chalcone synthase, dihydroflavonol 4-reductase and anthocyanidin synthase) and the accumulation of flavonoids and hydroxycinnamic acids were determined in response to E. splendidum infection. A pathogenesis-related (PR 4) gene was isolated and its expression was studied in host ramet leaves. To our knowledge, this was the first successful artificial infection reported with E. splendidum. Disease frequencies of the inoculated ramets were between 32% and 47% for E. splendidum and 33% for E. vaccinii, but below 10% in uninoculated control ramets. There were no differences in disease frequencies between V. vitis-idaea populations. Both symptomatic leaves and healthy leaves of diseased ramets showed activation of flavonoid biosynthesis at the gene level, whereas expression of PR 4 was observed only in symptomatic leaves. The increase of flavonoid biosynthesis in healthy leaves of diseased ramets may represent a general response to stress or a role in defence against the pathogen E. splendidium. Ability of V. vitis-idaea to defend chemically against Exobasidium fungi and the heterogeneity of genotypes, age, size, and growth rates in host plant populations might be reasons for the low infection incidence of Exobasidia in nature.     

Germination response of local Southern European populations of Datura stramonium at a range of constant temperatures

Studies on weed germination are required to develop efficient integrated weed management strategies that should be transferable at wide scale and accurate at local level. To study interpopulation variability, which may hamper this process, three local populations of Datura stramonium were collected in Italy, Portugal and Spain and then cultivated simultaneously in three common gardens under different environmental conditions. Hence, nine seed lots were included in the germination tests performed at a range of constant incubating temperatures. Significant effects of population of origin, cultivation site, incubating temperature and their interaction were detected on mean germination percentage and germination t₅₀. Mean germination percentage varied from 0.0 to 89.7% ± 2.10 SE at 8 and 20°C respectively. Mean germination t₅₀ ranged from 636.1 ± 24.86 SE to 267.0 ± 16.06 SE hours at 12 and 20°C respectively. Seed lots deriving from the Portuguese population presented the lowest germination percentages and highest germination t₅₀ at low incubating temperatures, regardless of the cultivation site. This behaviour could be related to an adaptive process of the Portuguese population to the local environmental conditions, produced by the combination of climate and agronomic management. The presence of high interpopulation variability for D. stramonium could hinder the development of transferable IWM strategies. However, management plans could be proposed at regional scale according to the approach of modifying the patterns of agronomic disturbances to which the local populations have adapted.     

Factors affecting seed germination and emergence of button grass (Dactyloctenium radulans) (R.Br.) P.Beauv.

Button grass (Dactyloctenium radulans) is a native, widely spread summer grass weed species in Australia. However, limited information is available on the seed germination biology of this species. Experiments were conducted to evaluate the effect of environmental factors on the germination and emergence of two populations of D. radulans. The seeds of these populations were collected separately from Dalby, Queensland and Coleambally, NSW. Seeds were germinated at a range of constant and alternating temperatures (25/15, 30/20, 35/25 and 40/30°C day/night). The greatest seed germination was at a constant temperature of 30°C. Seed germination was reduced at the lowest alternating temperature (25/15°C). Germination of both populations was strongly stimulated by light, suggesting a great amount of emergence of D. radulans on bare ground, such as crop seedbeds. Germination of the D. radulans population collected from a northern cotton farming system (Dalby) was more tolerant to a greater range of salt stress than the population sourced from the south (Coleambally). Seeds of both populations germinated over a wide range of pH, between 4 and 10. However, germination was the greatest in a high pH buffer solution, indicating that the species prefers to germinate in alkaline soil. These results showed that D. radulans seeds possess a wide range of tolerance mechanisms to different environmental stresses. Information obtained in this study will help in developing more sustainable and effective integrated weed management strategies for the control of this weed and weeds with similar responses in summer cropping systems, such as cotton.     

Het optreden van plaatselijke rassen bij Helopeltis antonii Sign. op Java

Summary Helopeltis antonii was bred at different constant temperatures at the experimental station at Semarang in 1939 and 1940. Some of the results concerning the duration of development from hatching of the egg untill the imaginal stage are recorded in the table and the graph. By comparing these data with those published byLeefmans (1916) (Helopeltis bred on tea in West Java) andZehntner (1901) (Helopeltis bred on cocoa in Central Java) we find that the time of development mentioned by these authors is shorter. In accordance with the experiments ofBonnemaison (1946) this probably can be attributed to the breeding ofHelopeltis at alternating temperatures (day and night). The duration of development at 19°C, mentioned byLeefmans (19 days), however, differs so much from the duration found in this investigation (32–34 days) that this explanation seems improbable. Other experiments at constant temperature, that will be published elsewhere, show that the population used for breeding was well adapted to the average temperature at the estate, Djati Rungo, from where this population came. This extreme adaptation suggests that this may also occur at other localities. Therefore it is probable that the populations ofH. antonii from different localities do not have the same genetical composition. The differences mentioned above can probably be attributed therefore to this fact. Data are given to strengthen this supposition. The existence of genetically different populations has already been suggested by ProfessorRoepke in 1909. He called them biological races. The occurrence of the varietybradyi ofH. antonii is probably caused by such a difference too and thus could better be considered as a subspecies.Helopeltis theobromae Mill. is without doubt only a subspecies ofH. theivora.Thus we suggest:Helopeltis antonii subspec.bradyi Wat. new comb. Helopeltis theivora subspec.theobromae Mill. new comb.

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Deventer     

Temperature-dependent development rates of <Emphasis Type="Italic">Bracon vulgaris,</Emphasis> a parasitoid of boll weevil

The duration of development of Bracon vulgaris Ashmead, parasitoid of the boll weevil Anthonomus grandis Boheman, was determined at nine constant temperatures between 18°C and 38°C. Nonlinear regression analysis was used to test the fit of temperature-dependent development rates to the Sharpe and DeMichele and Lactin et al. models. At the highest tested temperature (38°C) all the parasitoid eggs died before hatching and no evidence of development was observed. The high values of R ² for the models of Sharpe and DeMichele (0.8432 to 0.9834), and Lactin et al. (0.9071 to 0.9795) indicated that these models are suitable to estimate the development rate of B. vulgaris as a function of temperature. B. vulgaris showed tolerance to high temperature which is represented by the high value of H _H (change in enthalpy associated with high-temperature inactivation of the enzyme) for the prepupa stage of this insect obtained with the Sharpe and DeMichele model. According to that model, B. vulgaris exhibits thermal stress at 35.7°C, which indicates that maximum thermal stress estimated by this model was close to the real one.     

Phenotypic Differences Between vacuma and transposa subpopulations of Botrytis cinerea

One hundred and twenty-one single-spore strains of Botrytis cinerea isolated from Bordeaux vineyards were molecularly characterized as either transposa or vacuma, two subpopulations of B. cinerea distinguished by the presence of transposable elements. Forty-three vacuma and 68 transposa strains were distributed into two main classes (mycelial or sclerotial) by morphological phenotype according to the organ of origin. Strains isolated from overwintering sclerotia produced exclusively sclerotial colonies. The mycelial growth rate of 21 transposa and 13 vacuma strains was significantly influenced by agar-medium and temperature. The mycelial growth rate was significantly strain-dependent at favourable temperatures (15, 20 and 25 °C), but not at limiting ones (5 and 28 °C): vacuma strains showed the fastest growth rates. The strains of the two subpopulations were similar in virulence on both host species tested (Vitis vinifera and Nicotiana clevelandii). The grapevine leaves were significantly more susceptible to B. cinerea than those of tobacco. A significant negative correlation was established between virulence and mycelial growth rate. The epidemiological consequences concerning population structure of B. cinerea in vineyards are discussed.     

Survival of Xanthomonas campestris pv. campestris in the phyllosphere and rhizosphere of weeds

The phyllosphere and rhizosphere of weeds are important niches for phytobacterial survival. The absence of information in Brazil regarding Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, motivated this study. Twenty‐six weed species belonging to 14 botanical families were included in field experiments between August 2014 and October 2015. Lepidium virginicum and Raphanus raphanistrum (Brassicaceae) demonstrated great potential for survival of Xcc in the phyllosphere, with the bacterium isolated after 56 and 70 days, respectively. Low variation between maximum and minimum temperatures, high rainfall and high relative humidity at specific times of the year contributed to longer Xcc survival periods in the phyllosphere of some species. Xcc survived in the rhizosphere only in R. raphanistrum, where it was isolated for up to 28 days. No relation was found between climatic factors and survival in the rhizosphere. The data indicate that control of brassicaceous weeds will contribute to the control of black rot.     

Does soil warming affect the interaction between Pasteuria penetrans and Meloidogyne javanica in tomato plants?

A system to grow tomato plants infected by Meloidogyne javanica under constant temperatures of 18, 21, 24, 27 and 30 °C was developed and used to assess how temperature and the application of the biological control bacterium Pasteuria penetrans affected plant growth, the nematode population and endospore production. Each plant was inoculated with 300 second‐stage juveniles (J2) with four or five spores of P. penetrans attached to their cuticles or with 300 nematodes without P. penetrans. Increasing soil temperature increased tomato growth, the number of endospores per female, and the number of galls of M. javanica at the end of 38 days. Increasing temperatures up to 27 °C also increased the number of egg masses produced by M. javanica. Presence of P. penetrans reduced the numbers of galls and egg masses at all temperatures by up to 52.2% and 61.4% at 27 and 30 °C, respectively. Pasteuria penetrans reduced the M. javanica population even at soil temperatures of 18 and 21 °C. However, temperatures of 27 and 30 °C enhanced nematode control and the production of P. penetrans endospores is faster. The system developed in this work is simple and efficient for growing plants under constant temperatures and can be used for different purposes.     

Phytophthora boehmeriae boll rot: a new threat to cotton cultivation in the mediterranean region

A boll rot of cotton (Gossypium hirsutum L.) was observed for the first time in Greece in August 1993 in Larissa and Volos counties, and in August and September 1995 in Trikala and Phthiotis counties. Fungi of the genusPhytophthora were isolated from diseased plants. Morphological characteristics of the pathogen were recorded on mounts made directly from the infected tissues or after growth of the isolated fungus on corn meal agar or sterile distilled water. Colony morphology, growth rates, features of asexual and sexual structures and maximum growth temperatures were examined. APhytophthora species new to Europe,Phytophthora boehmeriae Sawada, attacking cotton bolls, was identified. The pathogenicity of the isolates was confirmed by artificial inoculations of detached cotton bolls. Analysis of α-esterase isozymes revealed unique banding patterns for isolates ofP. boehmeriae compared with those ofP. cactorum andP. parasitica, which arePhytophthora species with similar morphology.     

Multilocus sequence typing analysis of Italian Xanthomonas campestris pv. campestris strains suggests the evolution of local endemic populations of the pathogen and does not correlate with race distribution

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in Brassicaceae. It is widespread in Italy and severe outbreaks occur under conditions that favour disease development. In this study a multilocus sequence typing approach (MLST) based on the partial sequence of seven loci was applied to a selection of strains representative of the main areas of cultivation and hosts. The aim was to investigate whether the long tradition of brassica crops in Italy has influenced the evolution of different Xcc populations. All loci were polymorphic; 14 allelic profiles were identified of which 13 were unique to Italian strains. Based on the seven loci, the most common genotype within the Italian Xcc strains (AP1) was also the most representative genotype found in worldwide Xcc strains. This genotype was included in a new clonal complex in addition to three other clonal complexes already identified in Xcc populations. The phylogenetic reconstruction using a concatenated dataset of four conserved protein-coding genes, dnaK, fuyA, gyrB and rpoD, showed that the Italian strains belonged to two genetic groups. Physiological races were also investigated for the first time in Italy. The race structure of Xcc was determined by inoculating eight differential Brassica lines belonging to five species and showed that, in Italy, race 4 is the most widespread, followed by races 1 and 6. No correlation was found between allelic profiles, host of isolation, geographical origin and races, although a prevalent race was identified within the same clonal complex.     

Multiplication of Xanthomonas campestris pvs secalis and translucens in host and non-host plants (rye and barley) and development of water soaking1

Multiplication of the rye-specific and barley-specific pathovars secalis and translucens, respectively, of Xanthomonas campestris (earlier named Xanthomonas translucens f. sp. cerealis) was determined in two rye and two barley cultivars. Fresh bacterial cultures were suspended in boiled tap water, adjusted to about 3 × 10⁷ cfu ml^?1 and infiltrated into the mesophyll of young leaves. In the compatible combinations, a generation time of 1.7–2.1 h was determined. Significantly higher multiplication rates than in incompatible combinations were not observed before 3 or 4 days after inoculation. At this time, the first small water-soaked areas appeared. Bacteria multiplied up to the seventh day after inoculation, reaching a maximum of at least 10⁹ cells per cm², the same time at which the water-soaked areas had reached their maximum size. Thus, the rate of bacterial multiplication paralleled with the development of the water-soaked areas. In the incompatible combinations, nearly the same generation times were detected during the exponential growth phase. However, water-soaked areas did not appear, and the final bacterial concentration ranged from 1:1000 to 1:100 of that determined in compatible combinations. A distinct early lag phase, characterized by a decrease of the bacterial population down to 1:100 of the starting concentration, was detected in all cases.     

The effect of temperature on glutathione S-transferase activity and glutathione content in Alopecurus myosuroides (black grass) biotypes susceptible and resistant to herbicides

Herbicide‐resistant populations of Alopecurus myosuroides (black grass) have become widespread throughout the UK since the early 1980s. Previous observations in this laboratory have demonstrated that natural climatic fluctuations caused increases in endogenous glutathione S‐transferase (GST) enzyme activity in A. myosuroides plants as they mature, which is thought to be linked to herbicide resistance in this species. The present study has investigated the effects of plant growth at 10°C and 25°C, and reports GST specific activity and glutathione (GSH) pool size in resistant and susceptible A. myosuroides biotypes. Findings demonstrate differences in GST activity between resistant and susceptible populations, which are transient at lower growth temperatures. The GSH pool size was elevated at lower growth temperature in both biotypes. We speculate that these endogenous responses are part of a natural mechanism of acclimation to environmental change in this species and suggest that resistant plants are more able to adapt to environmental stress, as indicated in this instance by temperature change. These observations imply that the control of resistant A. myosuroides by graminicides may be more effective when applied at lower temperatures and at earlier growth stages.     

Differentiation of Xanthomonas campestris pv. Phaseoli from Xanthomonas campestris pv. Phaseoli var. Fuscans by PFGE and RFLP

Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, the causal agents of the common and fuscous bacterial blight of beans, appear to be phenotypically identical except that the latter can produce a melanin-like pigment in culture. Ten isolates of X. campestris pv. phaseoli and 12 isolates of X. campestris pv. phaseoli var. fuscans were examined using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP). The average genome sizes for X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were 3850.6±48.9 and 3584.3±68.1kb respectively. The genetic relatedness of the isolates was determined from macrorestriction patterns generated using XbaI. Cluster analysis indicated that the non-fuscous and fuscous strains are distinct. RFLP results, based on the highly conserved hrp genes and a pectate lyase gene from Xanthomonas, also indicated that the two bacteria are genetically different. The results obtained in this study suggest that this pathovar can be segregated into two subgroups under a recently proposed reclassification of the Xanthomonas genus.     

糖苷水解酶基因FvGH16-2参与拟轮枝镰孢菌生长发育及致病性

为明确糖苷水解酶16（glycoside hydrolase,GH16）家族基因在拟轮枝镰孢菌Fusarium verticillioides生长发育和致病过程中的作用,利用生物信息学分析软件对其GH16家族成员进行鉴定,采用转录组分析及实时荧光定量PCR技术对GH16家族成员在不同培养温度下的表达水平进行检测,并通过遗传转化法对家族成员FvGH16-2基因进行敲除及回补,分析其在拟轮枝镰孢菌生长发育及致病过程中的作用。结果显示,在拟轮枝镰孢菌基因组中共鉴定到23个GH16家族基因,其中FvCH16-2、FvCH16-4和FvCH16-12等多个上调表达基因与病菌的生长和抗逆性相关。敲除FvGH16-2基因导致拟轮枝镰孢菌的生长速率及产孢能力降低,细胞壁完整性受损,对H2O2更加敏感,同时对玉米籽粒和茎秆的致病力减弱。表明FvGH16-2基因在拟轮枝镰孢菌生长发育和致病过程中发挥着重要作用,是拟轮枝镰孢菌细胞壁完整性和致病性所必需的。     

Natural tolerance of Cuscuta campestris to herbicides inhibiting amino acid biosynthesis

The response of Cuscuta campestris Yuncker, a non‐specific above‐ground holoparasite, to amino acid biosynthesis inhibitor (AABI) herbicides, was compared with other resistant and sensitive plants in dose–response assays carried out in Petri dishes. Cuscuta campestris was found to be much more resistant to all AABI herbicides tested. The I₅₀ value of C. campestris growth inhibition by glyphosate was eightfold higher than that of transgenic, glyphosate‐resistant cotton (RR‐cotton). The I₅₀ value for C. campestris shoot growth inhibition by sulfometuron was above 500 μM, whereas that of sorghum roots was only 0.004 μM. Cuscuta campestris exposed to glyphosate gradually accumulated shikimate, confirming herbicide penetration into the parasite and interaction with an active form of the target enzyme of the herbicide, 5‐enolpyruvylshikimate‐3‐phosphate synthase. More than half of the C. campestris plants associated with transgenic, glyphosate‐resistant sugarbeet (RR‐sugarbeet) treated with glyphosate or with transgenic, sulfometuron‐resistant tomato (SuR‐tomato) treated with sulfometuron recovered and resumed regular growth 20–30 days after treatment. New healthy stems developed, followed by normal flowering and seed setting. The results of the current study demonstrate the unique capacity of C. campestris to tolerate high rates of AABI. The mechanism of this phenomenon is yet to be elucidated.