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Among European Neottieae, Limodorum abortivum is a common Mediterranean orchid. It forms small populations with a patchy distribution in woodlands, and is characterized by much reduced leaves, suggesting a partial mycoheterotrophy. We have investigated both the photosynthetic abilities of L. abortivum adult plants and the diversity of mycorrhizal fungi in Limodorum plants growing in different environments and plant communities (coniferous and broadleaf forests) over a wide geographical and altitudinal range. Despite the presence of photosynthetic pigments, CO2 fixation was found to be insufficient to compensate for respiration in adult plants. Fungal diversity was assessed by morphological and molecular methods in L. abortivum as well as in the related rare species Limodorum trabutianum and Limodorum brulloi. Phylogenetic analyses of the fungal internal transcribed spacer (ITS) sequences, obtained from root samples of about 80 plants, revealed a tendency to associate predominantly with fungal symbionts of the genus Russula. Based on sequence similarities with known species, most root endophytes could be ascribed to the species complex encompassing Russula delica, Russula chloroides, and Russula brevipes. Few sequences clustered in separate groups nested within Russula, a genus of ectomycorrhizal fungi. The morphotypes of ectomycorrhizal root tips of surrounding trees yielded sequences similar or identical to those obtained from L. abortivum. These results demonstrate that Limodorum species with inefficient photosynthesis specifically associate with ectomycorrhizal fungi, and appear to have adopted a nutrition strategy similar to that known from achlorophyllous orchids.

In 2018 there were over 1.8 million new cases worldwide of colorectal cancer and relapses after clinical treatments. Many studies ascribe the risk of the appearance of this cancer to the Western life style : a sedentary life, obesity, and low -fiber, high -fat diets can promote the onset of disease. Several studies have shown supplement phytochemicals to have an inhibiting effect on the growth of various cancers through the activation of apoptosis. Our goal was to prove the effectiveness of a natural compound in the combined therapy of colorectal cancer. Trigno M supplement was an optimal candidate as anticancer product for its high concentrations of phenolic acids, flavonoids and anthocyanins. Our work showed the antitumor activity of Trigno M, extract of Prunus spinosa drupes combined with the nutraceutical activator complex (NAC), in 2D, 3D and in vivo colorectal cancer models. The cellular model we used both in vitro and in vivo was the HCT116 cell line, particularly suitable for engraftment after inoculation in mice. Trigno M inhibited the growth and colony formation of HCT116 cells (35%) as compared to the chemotherapy treatment with 5-fluorouracil (80%) used in clinical therapy. The reduction of the morphological dimensions in the spheroid cells after Trigno M, was compared with 5-fluorouracil demonstrating the efficacy of the Trigno M compound also in 3D models. Flow cytometric analysis on 3D cells showed a significant increase in the apoptotic cell fraction after Trigno M treatment (44.8%) and a low level of necrotic fraction (6.7%) as compared with control cells. Trigno M and 5-fluorouracil induced the apoptosis in a comparable percentage. Monotherapy with Trigno M in severely immunodeficient mice, carrying colon rectal cancer xenografts, significantly reduced tumor growth. The histopatological analysis of the ectopic tumors showed a lower level of necrosis after Trigno M treatment compared with the control. We conclude that Trigno M is well tolerated by mice, delays colorectal cancer growth in these animals and should be weighed up for integration of the current multi-drug protocols in the treatment of colon carcinoma.

ADDITIONAL INDEX WORDS. Capsicum annuum, fluorescence, gas exchange, photosynthesis, CO 2 transfer conductance, vegetative and fruit biomass, drought stress ABSTRACT. Physiological characteristics, growth, and biomass production of rainfed and irrigated bell pepper ( Capsicum annuum L. var. anuum (Grossum Group) 'Quadrato d'Asti') plants were measured in the semiarid conditions of a Mediterranean summer to determine if drought stress effects are transient and do not affect plant growth and crop yield or are persistent and adversely affect plant growth and crop yield. A low midday leaf water potential indicated the occurrence of transient drought stress episodes in rainfed plants during the first 2 months of the study. Later on, predawn water potential also increased, indicating a persistent drought stress condition despite the occurrence of some rainfall. Photosynthesis was reduced when stress conditions developed, but the reduction was transient and limited to the central part of the day during the first 2 months. As plants aged, however, the impact of drought stress on photosynthesis was not relieved during the overnight recovery period. Stomatal conductance was reduced both during transient and permanent stress conditions while CO2 transfer conductance (i.e., conductance to CO 2 inside the leaf) was only reduced when photosynthesis inhibition was unrecoverable. However, chloroplast CO 2 concentration was higher in rainfed than in irrigated leaves indicating that CO 2 availability was not limiting photosynthesis. Nonphotochemical quenching of fluorescence increased significantly in rainfed leaves exposed to permanent stress indicating the likely impairment of ATP synthesis. Transient inhibition of photosynthesis did not significantly affect leaf area index and biomass production, but growth was significantly reduced when photosynthesis was permanently inhibited. Fruit dry weight was even higher in rainfed plants compared to irrigated plants until drought stress and photosynthesis reduction became permanent. It is suggested that bell pepper growth without supplemental irrigation over the first part of the vegetative cycle does not impair plant growth and may even be useful to improve yield of early fruit.

Short-term fumigation with 1% methanol in air was carried out to investigate effects on the photosynthetic apparatus of horticultural species characterized by leaves with different stomatal distribution. Methanol decreased the photosynthetic capacity of all species. The hypostomatous cherry (Prunus avium L.) was the most sensitive species. Between the two amphistomatous species, the effect was smaller in pepper (Capsicum annuum L. var. annuum) than in melon (Cucumis melo L.). A 4-minute fumigation caused a stronger inhibition of photosynthesis than a 90-second fumigation. The time course of the inhibition of the photosynthetic electron transport following a methanol fumigation of cherry leaves suggests that methanol starts inhibiting photosynthesis and photorespiration after ≈60 seconds and that the effect is complete after 180 seconds. This inhibition is not permanent, however, since gas-exchange properties recovered within 24 hours. Methanol vapor effects were greatest when leaves were fumigated on the surfaces with stomata. However, fumigation with methanol does not affect stomatal conductance. Therefore, inhibition of photosynthesis following methanol fumigation can be attributed to a temporary inhibition of biochemical reactions. reports show that methanol does not affect the CO2 compensation point (Poskuta et al., 1995, van Iersel et al., 1995), an indication that photorespiration is not preferentially suppressed by methanol. Simultaneous measurements of gas exchange and chlorophyll fluorescence, allow one to partition the electron transport rate between photosynthesis and photorespiration (Di Marco et al., 1993). We used this approach to accomplish our second goal, that is, to understand if methanol vapor rapidly affected biochemistry of photosynthesis and selectively inhibited photorespiration. It is possible that the different results obtained with methanol sprays reflect differences in the exposure and acquisition of methanol by the leaf. Under field conditions these cannot be controlled accurately. For instance, acquisition may be inefficient because of temperature-dependent volatilization of the methanol solution. The third goal of this study was to eliminate these uncertainties while investigating methanol effects on stomata and biochemistry. We used gaseous methanol in a totally controlled environment. This eliminated the variability of environmental factors and allowed us to study a direct effect of methanol vapor rather than an effect that may depend on the environment-controlled evolution of methanol vapor from sprays. Materials and Methods Seedlings of pepper (Capsicum annuum ‘Quadrato d’Asti’) and melon (Cucumis melo ‘Supermarket’) were grown for ≈2 months in 3-dm pots. Two-year-old cherry (Prunus avium ‘Gisella’) trees were grown in 15-dm pots. Soil was a mixture of 1 sand : 1 peat : 1 lime. Pots were regularly watered to soil saturation and weekly fertilized according to the Ingestad and Agren (1992) method. Plants were grown under conditions typical of summer in the experimental field of IBEV (Rome, Italy 41° 50' latitude). Daily light intensity and air temperature varied between 700 to 2000 μmol·m·s and 25 to 37 °C, respectively, depending of sky cloudiness. The experiments were conducted on the last fully expanded leaf. The leaf was enclosed in a gas exchange cuvette with 4.9-cm Foliar sprays of methanol solutions were reported to increase the yield of crops (Nonomura and Benson, 1992). Other experiments confirmed (Valenzuela et al., 1994) or disproved (Albrecht et al., 1995; Faver and Gerik, 1996; McGiffen et al., 1995; van Iersel et al., 1995) that methanol has a beneficial effect on crop yield. Yield improvement was especially noticed in C3 plants growing in arid, high sunlight environments but was not observed in C4 plants (Nonomura and Benson, 1992). Apparently, the leaf turgor of methanol-sprayed leaves was higher than in nonstressed controls. This led to speculation that methanol spraying increased stomatal conductance, in turn resulting in improved carbon acquisition (Nonomura and Benson, 1992). However, Nonomura and Benson (1992) did not report any direct measurements of CO2 and H2O exchange between leaves and air. More recently, methanol was found to improve photosynthesis and stomatal conductance but not growth of cotton (Faver and Gerik, 1996) and was ineffective on sour orange (Idso et al., 1995) and on fescue (Poskuta et al., 1995). A first goal of our work was to understand how stomata are affected by methanol. We investigated the effect of methanol on horticultural plants with different stomatal distribution as estimated by exchange of water vapor in a gas exchange cuvette. We used one hypostomatous species (cherry) and two species amphistomatous but with stomata prevalently distributed on the abaxial (pepper) and on the adaxial (melon) leaf surface, respectively. This approach also allowed us to investigate if methanol penetration occurred through stomata and/or cuticle and if the methanol effect on photosynthesis was mediated by changes of stomatal opening. Nonomura and Benson (1992) also speculated that an inhibition of photorespiration could improve carbon fixation of C3 plants exposed to methanol sprays. This would have explained why the effect was not observed in C4 plants. Gas exchange-based

On a etudie en serre la formation des feuilles de plants de mais en pot soumis a des stress hydriques severes. Le cycle de stress hydrique a commence au debut du stade vegetatif (stade cinq feuilles). Les plants temoins et les plants stresses ont recu respectivement 100 et 50 % de l'eau d'evapotranspiration. Dix jours plus tard la moitie des plants subissant le stress hydrique ont ete irrigues au meme niveau que les temoins. Le stress hydrique a diminue le potentiel osmotique de toutes les feuilles le long du plant, ainsi que leur concentration en chlorophylle et le taux de photosynthese. La surface de la feuille des plants stresses etait reduite, tandis que la concentration des feuilles en azote etait plus elevee que chez les temoins a la fin du stade vegetatif. Apres avoir ete a nouveau arroses, les plants'ont recupere et leur taux de photosynthese foliaire ainsi que la turgescence des feuilles ont augmente, tout en restant inferieurs a ceux des temoins. A la fin du stade vegetatif, la concentration en azote des feuilles des plants re-arroses etait semblable a celles des plants stresses, mais la croissance foliaire n'avait pas repris bien que le taux relatif de mort soit descendu au niveau de celui des plants temoins. Ceci suggere que les plants ayant subi un stress hydrique severe sont capables, lorsqu'ils sont arroses a nouveau, de bien retablir les processus physiologiques, a un moindre niveau toutefois que celui des temoins parce que la concentration en chlorophylle n'est pas totalement recuperee.

Water scarcity is one of the most detrimental effects on crop yield. We studied the effect of water stress on carbon acquisition and allocation to monoterpenes of commercial importance in spearmint and rosemary plants. Increasing levels of water stress reduced photosynthesis and plant biomass. Under increasing water-stress levels photosynthesis was limited by low CO2 availability due to reduced stomatal and mesophyll conductance, while photochemical and biochemical processes were not affected. At the balsamic period, significantly higher amounts of monoterpenes per unit mass were formed in water-stressed leaves with respect to irrigated controls. However, because of plant biomass reduction, water stress reduced the total yield of monoterpenes. Appropriate irrigation strategies may, therefore, potentially improve yield of monoterpenes in field grown spearmint and rosemary crops.

Scarce water resources frequently limit crop production in semi-arid lands. Drip irrigation may constitute a method for sagacious management of water resources, but there is still limited information on its use for sugar beet crop in Mediterranean environments. The present study was conducted to determine the effect of irrigation on sucrose accumulation and root yield, as well as on sugar beet physiological responses, and to compare drip and low-pressure sprinkler (high efficiency) irrigation performances for sugar quality. As the main scope of the study was to give indications to allow water saving, the comparison focused on the best irrigation method when a similar amount of water was applied. Sugar beets were grown in an experimental field site in southern Italy (Molise Region) for two consecutive growing seasons (1999/2000 and 2000/2001). The water applied ranged from 50, 75, and 100% of the estimated evapotranspiration (ET); unirrigated sugar beets were taken as control plants. In both years, harvest was completed in two periods. Increasing volume of water replaced was advantageous for crop performances, and yield and physiological responses of sugar beets drip irrigated with 75% of estimated ET matched in most cases those of low-pressure sprinkler irrigated with 100% of estimated ET, resulting in potential 25% of water volume saving. In general, drip irrigation influenced positively many of the considered physiological and technological parameters, as compared to low-pressure sprinkler irrigation. Differences in root production and sugar quality between the two harvest periods were not significant. Results indicate that (1) increasing the amount of water applied gives benefits in terms of sugar beet root yield and sucrose accumulation, (2) drip irrigation (even applied every-other-furrow) appears to be consistently advantageous with respect to low-pressure sprinkler irrigation for sugar beet performances in semi-arid environments, (3) there are indications that suggest sugar beet growers should harvest in advance (of traditional harvest dates).

Eucalyptus spp. are among the highest isoprene emitting plants. In the Mediterranean area these plants are often cultivated along the seashore and cope with recurrent salt stress. Transient salinity may severely but reversibly reduce photosynthesis and stomatal conductance of Eucalyptus globulusleaves but the effect on isoprene emission is not significant. When the stress is relieved, a burst of isoprene emission occurs, simultaneously with the recovery of photosynthetic performance. Later on, photosynthesis, stomatal conductance, and isoprene emission decay, probably because of the onset of leaf senescence. Isoprene emission is not remarkably affected by the stress at different light intensities, CO2 concentrations, and leaf temperatures. When CO2 was removed and O2 was lowered to inhibit both photosynthesis and photorespiration, we found that the residual emission is actually higher in salt-stressed leaves than in controls. This stimulation is particularly evident at high-light intensities and high temperatures. The maximum emission occurs at 40°C in both salt-stressed and control leaves sampled in ambient air and in control leaves sampled in CO2-free and low-O2 air. However, the maximum emission occurs at 45°C in salt-stressed leaves sampled in CO2-free and low-O2 air. Our results suggest the activation of alternative non-photosynthetic pathways of isoprene synthesis in salt-stressed leaves and perhaps in general in leaves exposed to stress conditions. The temperature dependence indicates that this alternative synthesis is also under enzymatic control. If this alternative synthesis still occurs in the chloroplasts, it may involve a thylakoid-bound isoprene synthase.

Salt accumulation in spinach (Spinacia oleracea L.) leaves first inhibits photosynthesis by decreasing stomatal and mesophyll conductances to CO2 diffusion and then impairs ribulose-1,5-bisphosphate carboxylase/oxygenase (S. Delfine, A. Alvino, M. Zacchini, F. Loreto [1998] Aust J Plant Physiol 25: 395–402). We measured gas exchange and fluorescence in spinach recovering from salt accumulation. When a 21-d salt accumulation was reversed by 2 weeks of salt-free irrigation (rewatering), stomatal and mesophyll conductances and photosynthesis partially recovered. For the first time, to our knowledge, it is shown that a reduction of mesophyll conductance can be reversed and that this may influence photosynthesis. Photosynthesis and conductances did not recover when salt drainage was restricted and Na content in the leaves was greater than 3% of the dry matter. Incomplete recovery of photosynthesis in rewatered and control leaves may be attributed to an age-related reduction of conductances. Biochemical properties were not affected by the 21-d salt accumulation. However, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and content were reduced by a 36- to 50-d salt accumulation. Photochemical efficiency was reduced only in 50-d salt-stressed leaves because of a decrease in the fraction of open photosystem II centers. A reduction in chlorophyll content and an increase in the chlorophylla/b ratio were observed in 43- and 50-d salt-stressed leaves. Low chlorophyll affects light absorptance but is unlikely to change light partitioning between photosystems.

We analyzed the effect of NaCl stress on photorespiration of spinach leaves by calculating the rate of carboxylation/oxygenation of ribulose-1,5-bisphosphate carboxylase/oxygenase, and by measuring the content of amino acids produced through photorespiration. After 20 d of NaCl stress the carboxylation rate was reduced while the oxygenation rate was not affected. The contents of serine, glycine, and alanine increased relevantly. The amount of glutamine also increased after 20 d but the amount of glutamate did not. Hence photorespiration may be stimulated under moderate NaCl stress. A relevant electron transport rate was observed under CO2-free air, which may indicate refixation of photorespiratory CO2. When NaCl accumulation proceeded for more than 20 d, photosynthesis was reduced and the content of photo-respiratory amino acids started to decrease, but the oxygenation rate did not change.

Photorespiration rate can be estimated in vivo but the actual amount of photorespiratory CO2 emitted or recycled by the leaf is largely unknown. We exploited the insensitivity of infrared gas analy-zers to 13CO2 to detect the photorespiratory CO2 emission of leaves exposed to air containing only 13CO2. Photorespiratory CO2 emission was calculated by subtracting the 12CO2 emitted under non-photorespiratory (2% O2) conditions from that emitted under photorespiratory (21% O2) conditions. Illuminated leaves of herbaceous and tree species emitted a constant amount of 12CO2 within 30–60 s after switching to 13CO2. Photorespiratory CO2 emission was less than 20% of the photorespiratory rate simultaneously calculated by combining fluorescence and gas exchange. Thus, the leaves recycled more than 80% of photorespiratory CO2. The highest recycling was associated with high rates of photosynthesis in tomato and spinach. In the dark, mitochondrial respiration measured by the emission of 12CO2 in air with 13CO2 was similar to that measured by conventional gas exchange in ambient air, thus confirming the accuracy of our method. In the light, mitochondrial respiration estimated from the emission of 12CO2 after complete labeling of photorespiration was lower than in the dark, suggesting either light-inhibition or recycling of respiratory carbon.