African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 5, Num. 2, 1997, pp. 189-195

The determination of when maize plants were infected with maize streak virus from the position of the lowest diseased leaf

R.W. GIBSON and W.W. PAGE^1

Namulonge Agricultural and Animal Production Research Institute, P.O. Box 7084, Kampala, Uganda
^1 Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent, ME4 4TB, UK

(Recieved 11 February, 1997; accepted 5 June, 1997)

Code Number: CS97024
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ABSTRACT

Maize plants cv Kawanda Composite A growing in soil in a screenhouse and ranging in age from 1 - 9 weeks were inoculated with maize streak geminivirus (MSV) using viruliferous Cicadulina mbila adults caged either in groups of two or five per plant on top, middle or bottom leaves in order to determine whether a consistent relationship is present between symptom development and plant age. Symptoms generally developed first on the leaf emerging at the time of inoculation or, for older plants, on the leaf directly below. Leaves which had already emerged did not subsequently develop symptoms, so the lowermost leaf with symptoms indicated the growth stage at which a plant was infected. This relationship allows the stages at which different plants in maize crops were infected with MSV to be determined from just a few surveys, so facilitating epidemiological studies at sites which can be monitored only infrequently, such as distant ones or farmers' fields.

Key Words: Cicadulina mbila, Kawanda Composite A, MSV, symptoms

RESUME

Les plantes de mais du cultivar Kawanda Composite A plantes en sol dans une serre, variant de 1 a 9 semaines d'age ont ete inocules avec la "streak" geminivirose du mais en utilisant des adultes virulents de Cicadulina mbila en cage en groupe de deux ou de cinq par plante, au sommet, au centre ou bien a la base foliaire. Generalement, les sympt“mes se sont developpes en premier sur la feuille qui a emerge au moment de l'inoculation ou sur la feuille directement en dessous de la precedente pour les plantes plus agees. Les feuilles qui avaient deja emerge n'ont pas developpe de sympt“mes, ainsi la feuille plus a la base avec sympt“mes a indique le stade de croissance a laquelle la plante a ete infectee. Ceci permet ainsi la determination, avec quelque peu d'enquetes, des stades auxquelles les plantes de culture de mais auraient ete infectes avec la virose du mais, devenant ainsi une methode appropriee pour l'etude de l'epidemiologie de la maladie en champs des fermiers ou pour des sites lointains.

Mots Cles: Cicadulina mbila, Kawanda Composite A, MSV, symptomes

INTRODUCTION

Maize streak geminivirus (MSV) is the most important viral pathogen of maize in sub-Saharan Africa, and is considered to be second only in importance in the region to African cassava mosaic geminivirus as a viral pathogen of crops (Geddes, 1990). Diseased plants are characterised by broken to almost continuous, longitudinal chlorotic streaks along the leaf surface; severely diseased plants may appear very pale green or white from a distance, are stunted and yield poorly (Storey, 1925; Pinner et al., 1988; Thottappilly et al., 1993). MSV is not seed-transmitted and infection is the result of transmission by leafhoppers belonging to certain species of the genus Cicadulina (Rose, 1978) including C. mbila as shown in the classical work by Storey (1925; 1928). Lower leaves produced before infection has occurred are symptomless (Storey, 1925) and symptoms develop only on new growth (Storey, 1936; Rose, 1978). Thus, the older a plant is when it is infected, the more disease-free lower leaves there are on that plant. Moreover, when upper leaves are inoculated, MSV antigens are detected only in young leaves produced above the inoculation point (Peterschmitt et al., 1992). The severity of maize streak disease (MSD) and the effect on yield both diminish the later an infection occurs during plant growth (Van Rensburg, 1981; Mzira, 1984).

The development of symptoms only on new growth produced after infection has occurred may allow the age of a plant at the time of infection to be estimated (Rose, 1978). This would allow the stages at which different plants in a crop were infected to be estimated by a few examinations made during a crop's development rather than by frequent, regular monitoring for the first appearance of disease on individual plants. This latter approach itself requires a variable correction factor being included to allow for the increasing time between inoculation and symptom appearance as plants age (Storey, 1928). The development of MSD is also affected by the numbers of leafhoppers used for inoculations (Storey, 1928; Van Rensburg et al., 1991), the MSV isolate (Pinner et al., 1988), the tolerance of the maize genotype (Efron et al., 1989) and temperature (Storey, 1928: Van Rensburg et al., 1991).

This study was done to test whether the linkage between time of infection and symptom appearance on the leaf developing at that time is predictable over a wide range of plant ages and inoculation conditions. If so, this would then provide an effective tool for monitoring the stage at which maize plants were infected.

MATERIALS AND METHODS

All experimental work was done with the local, open-pollinated maize cultivar Kawanda Composite A (KWCA) in a large screenhouse covered with "Nikofence 36" netting (Clovis-Lande Associates Ltd, Kent, TN12 5HH, UK) to exclude most small insects including Cicadulina. The maize was grown in the original soil over which the screenhouse was erected. Experiments were done during the first (Experiment 2) or second (Experiment 1) rains and received only natural rainfall. The MSV used in experiments was originally from naturally-infected field plants of KWCA showing typical MSD; fresh isolates were obtained for each experiment. Recordings of maximum and minimum outside shade air temperatures were taken daily.

Experiment 1: the effects of age of inoculated plants and numbers of viruliferous C. mbila on disease development.

Seed was sown on 16 September, 21 October and 11 November 1993 to obtain plots of plants of three different ages. Plots were in a randomised design of four blocks each of six plots, two planted on each of the sowing dates. In each plot, seed was sown to give two rows each of five plants; rows and plots were 0.9 m apart and plants 0.3 m apart along rows. Plants were inoculated by confining C. mbila raised on MSV-infected plants to the uppermost, readily accessible leaf using a clip cage. Either two or five adults were confined in a clip cage on 21 November for two days, so inoculation occurred when the differently-aged plants in each plot had emerged for either 1, 4 or 9 wks. Plants were sprayed with Dimethoate insecticide immediately after removing the adults to kill any hatching nymphs. Leaves on each plant were numbered using an indelible ink and the presence of symptoms on each was recorded daily. Overall MSD severity on plants was recorded on a 1-5 severity scale (1 = symptoms just visible; 5 = almost complete bleaching of leaves and severe stunting) at 8 day intervals. Observations ceased on 15 December when many plants reached the screenhouse roof.

Experiment 2: the effects of inoculating different leaves on development of MSD on plants of different ages.

Seed was sown on 25 May and 15 June 1994 to obtain plots of plants of two different ages. Plots were in a randomised design of four blocks each of four plots, two planted on each sowing date. In each plot, seed was sown to give two rows each of four plants, with 0.75 m between rows and 0.5 m between plants along rows. Plants were inoculated using two viruliferous C. mbila adults from 3 July for 2 days, approximately 1 or 4 wk after plant emergence. In each plot, two randomly-selected plants were inoculated with C. mbila confined to the top leaf, the middle leaf or the lowest surviving bottom leaf and two plants were not inoculated. Again, plants were subsequently sprayed with Dimethoate. Leaves were numbered and disease development monitored as before.

In both experiments, time to symptom development were compared using Student's t-test and proportions of plants developing MSD or MSD of particular severities were compared using Chi-squared test. Means are given + the standard deviation of individual observations.

RESULTS

Experiment 1: the effects of age of inoculated plants and numbers of viruliferous C. mbila on disease development.

Most plants developed MSD irrespective of the number of C. mbila used to inoculate each plant or the age of the plant inoculated (Table 1). However, the time to develop MSD increased from 3-4 days for 1 wk-old plants to 12-13 days for 9 wk-old plants, and there was a corresponding increase in the variability of the time to become diseased. Initial symptoms on the youngest inoculated plants comprised lines of white spots lying alongside the midrib and largely towards the base of the leaf. This generally occurred on the leaf which had been emerging when the plant was inoculated (Table 2). This first leaf to be diseased generally remained the lowest diseased leaf on a plant (Table 3). Symptoms on subsequent leaves to emerge were generally very severe, almost the entire leaf area being chlorotic, although symptoms became less severe on a few plants after 3-4 wks (Table 4). Symptoms on the first diseased leaf of most 4 and 9 wk-old inoculated plants appeared as pale streaks each perhaps 10 mm or more long and 1-2 mm wide lying within and along the midrib itself and, as with the 1 wk-old plants, again largely in the basal portion. Symptoms on subsequent diseased leaves comprised lines of smaller chlorotic streaks on and along the main leaf lamina itself rather than the midrib, and were again concentrated in the basal portion. Nine week old inoculated plants were already tall when inoculated (Table 1) and some had produced their full complement of leaves and begun to tassel; on these latter plants, streaking on the main stem itself was often the only symptom observed. As with 1 wk-old inoculated plants, most 4 wk-old inoculated plants developed symptoms first on the leaf emerging at the time of inoculation but on 9 wk-old inoculated plants it was generally the leaf below this that showed the first MSD (Table 3). Maize streak disease was generally moderate on these older plants, especially on 9wk-od inoculated plants, although on both 4 and 9 wk-old inoculated plants, disease gradually became more severe with time and plants inoculated with five C. mbila often had more severe symptoms than those inoculated with two (Table 4).

TABLE 1. Experiment 1. Presence of MSD on maize plants inoculated at different ages

-------------------------------------------------------------------------- 
Age of inoculated plant       1                4                9
       (weeks)        ----------------  ---------------  -----------------
Leafhoppers/plant        2        5        2       5        2        5
-------------------------------------------------------------------------- 

Diseased plants         35**     36**      39      40       36       38
Symptomless plants    0 (0%)   0 (0%)   1 (3%)   0 (0%)   4 (10%)   2 (5%)
Days to symptom 
appearance           4+/-1.4  3+/-0.6  6+/-2.3  7+/-3.1  12+/-4.9  13+/-6.3
  
Plant height (mm)*      147+/-44.8        483+/-96.4         970+/-174
Mean leaf number*       2.9+/-0.51        7.5+/-0.86        11.5+/-1.9
Max. temperature         31+/-2.4 C        31+/-1.6 C         29+/-2.3 C
Min. temperature         18+/-0.9 C        17+/-0.9 C         16+/-1.4 C
  

TABLE 2. Experiment 1. Concurrence between the first leaf to develop symptoms and the leaf appearing at the time of inoculation

-----------------------------------------------------------  
Age of inoculated plant     1           4           9
     (weeks)            ---------   ---------   ----------
Leafhoppers/plant        2     5     2     5      2     5    
-----------------------------------------------------------
Exact match (%)         74    83    63    68     33    20    
Leaf below* (%)         11     3    24    20     57    73    
Leaf above** (%)        14    14     8    10      3     0    
  

TABLE 3. Experiment 1. Concurrence between the first leaf to develop symptoms and the lowest leaf to be diseased at the end of the experiment

------------------------------------------------------------  
Age of inoculated plant      1            4           9
      (weeks)            ----------  -----------  ----------
Leafhoppers/plant         2     5      2     5      2     5
------------------------------------------------------------  
Exact match (%)          94    94     95    95      93    97
One leaf below (%)        6     6      5     5       7     3
------------------------------------------------------------

TABLE 4. Experiment 1. Changes with time in the proportions of plants with particular severities for plants inoculated with different numbers of leafhoppers and at different plant ages

-----------------------------------------------------------------------
Plants inoculated     2 leafhoppers/plant           5 leafhoppers/plant
      with:-        ---------------------           -------------------
Symptom severity     1   2   3   4   5   Chi-square   1   2   3   4   5
  score
------------------------------------------------------------------------  
Age*   Day**
1 wk   Day 8         0   8  21  58   13     n.s       0   3  30  66    0
        16           0   0   0   0  100     n.s.      0   0   0   0  100
        24           0   0   0  16   84     n.s.      0   0   0  11   89
  
4 wk   Day 8        50   47   3   0   0     n.s.     63  29   8   0    0
          16         8   24  29  39   0     n.s       3  15  38  34   11
          24         0   18  18  55   8    0.005%     3   3  26  42   32
  
 9 wk  Day 8       100    0   0   0   0     n.s.    100   0   0   0    0
          16        90   10   0   0   0    0.05%     66  14  21   0    0
          24        92    8   0   0   0    0.005%    64  22  14   0    0
  

Experiment 2: the effects of inoculating different leaves on development of MSD on plants of different ages.

As in Experiment 1, most plants inoculated via the top or middle leaves became diseased irrespective of plant age whereas most did not become diseased when the plants had been inoculated via bottom leaves (P<0.001) (Table 5). Nevertheless, the time taken for symptoms to appear was similar irrespective of inoculation site. As in Experiment 1, symptoms generally took longer to appear on the 4 wk-old than on the 1 wk-old inoculated plants (P<0.005). Irrespective of whether plants were inoculated on the top, middle or bottom leaf, symptoms again first appeared mostly on either the leaf which had been emerging at the time of inoculation or the leaf immediately above or below it (Table 6).

TABLE 5. Experiment 2. Effect of inoculating different leaves on the development of MSD

--------------------------------------------------------------------------- 
Age of plants                 1 wk                       4 wk
                  ----------------------------   --------------------------
Leaf inoculated     Top      Middle    Bottom     Top    Middle   Bottom
--------------------------------------------------------------------------- 
Plants diseased (of
 16 inoculated)   12 (75%)  14(88%)   6(38%)    10(63%)  13(82%)   3(18%)
Days to symptom 
  development     7+/-1.5   7+/-1.9  10+/-3.2  12+/-4.3  10+/-2.0  10+/-1.4
  
Max. temperature          26+/-1.1 C                    27+/-1.2 C
Min. temperature          16+/-1.1 C                    17+/-1.3 C
---------------------------------------------------------------------------

TABLE 6. Experiment 2. Concurrence between the first leaf to develop symptoms and the leaf appearing at the time of inoculation

------------------------------------------------------------------------- 
Age of inoculated plant        1 wk                     4 wk
                     -------------------------   ------------------------
Leaf inoculated       Top    Middle    Bottom     Top    Middle   Bottom
-------------------------------------------------------------------------  
Exact match           11       13         3        5        6        1
Leaf below             0        0         0        1        6        2
Leaf above             1        1         3        2        1        0
-------------------------------------------------------------------------

DISCUSSION

There was a very close relationship between the leaf that first developed MSD and the leaf stage when the plant was inoculated. When young plants (1 - 4 wks) were inoculated, the leaf first showing disease was almost always the one that was emerging at the time inoculation occurred as observed by Storey (1928, 1936), whereas with older plants, it was generally the next leaf down (Table 2). This relationship was only slightly affected by the number of viruliferous Cicadulina used (Table 2). Similarly, Storey (1928) and Van Rensburg et al. (1991) showed that symptoms appeared slightly quicker on plants inoculated with more Cicadulina. Disease was also slightly more severe when more Cicadulina were used (Table 4), as found by Van Rensburg et al. (1991) but not by Storey (1928). Site of inoculation also had little effect on symptoms, although it did have a marked effect on the probability of successful inoculation (Table 6).

The results also confirmed that symptoms developed within growing leaves (Storey, 1925) and did not appear on leaves which were already mature at the time of inoculation (Peterschmitt et al., 1992). The leaf which was first diseased generally remained as the lowest diseased leaf, acting as a marker until it senesced, although on a very small proportion of plants (Table 3) symptoms appeared in the basal region of the leaf below this first diseased leaf, probably as a result of continued activity of the cambium layer there. As a result of the consistent relationship between the leaf that first develops MSD and the leaf stage when a plant was inoculated, this confirms that the leaf stage when different plants in a crop were inoculated can be estimated (Rose, 1978) from a few surveys done during the growing season. Since individual plants develop more than one leaf per week, especially during the initial period of growth (Table 1) when infection is economically most damaging, the method is potentially as accurate as weekly monitoring of disease appearance on plants. However, the particular advantage of the method is that it allows the seasonal progress of infection to be monitored where only limited sampling may be possible, for example, in farmers' fields or at distant sites.

It was confirmed that on older plants symptoms were less severe (Table 4) (Van Rensburg, 1981; Mzira, 1984) and sometimes restricted to just the stem if leaf growth had finished. In addition, the time taken for symptoms to develop increased markedly with plant age, roughly doubling between 1 and 4 wk old plants in both experiments and again between 4 and 9wk old plants in Experiment 1. However, there was little evidence for any increase in resistance to becoming infected as the maize became older during the nine week period examined. This was surprising as it occurs in many other crop/virus combinations (e.g., Gibson, 1991) and has also been reported previously for MSV in maize (Storey, 1928).

The maximum air temperature was about 4 C higher and the minimum temperature about 1 C higher during symptom incubation for Experiment 1 than for Experiment 2, and symptom development for plants of similar ages took about twice as long in Experiment 2. This is in accord with previous experimental results showing that symptom appearance is delayed at lower temperatures (Demsteegt, 1984; Van Rensburg et al., 1991). This result emphasises the need to correct for the effect of temperature on the symptom incubation period when using regular monitoring of the first appearance of disease on individual plants in a crop to determine the time of inoculation as a change of only a few degrees changed the incubation period by several days. By contrast, the linkage between the appearance of disease on the leaf emerging at the time inoculation occurred was similar in both experiments, suggesting that it is relatively less affected by environmental conditions.

In summary, therefore, the two experiments have confirmed the suggestion by Rose (1978) that the stage at which a maize plant was infected can be estimated from a knowledge of the lowest diseased leaf on the plant and have shown that the relationship is robust, apparently little affected by vector numbers, environmental conditions, leaf inoculated or plant age.

ACKNOWLEDGEMENTS

This work was jointly undertaken under a scientific arrangement between the Ugandan National Agricultural Research Organisation and the Natural Resources Institute, UK. This publication is an output from the British Department for International Development of the United Kingdom, RNRRS-funded project A0526. The DFID can accept no responsibility for any information provided or views expressed. We wish to acknowledge valuable advice from Dr JM Thresh and technical support by Mr I Mpembe.

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Copyright 1997 The African Crop Science Society